Contrast agents

ABSTRACT

The invention provides a composition of matter of the formula (I): V-L-R, where V is a vector moiety having affinity for an angiogenesis-related endothelias cell receptor, L is a linker moiety or a bond and R is a detectable moiety, characterised in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species providing a multiplicity of labels detectable in in vivo imaging.

This application is a continuation application of U.S. application Ser.No. 09/422,977, filed Oct. 22, 1999, now U.S. Pat. No. 6,610,269 (ofwhich the entire disclosure of the pending, prior application is herebyincorporated by reference), for which the Issue Fee was paid on Jun. 17,2003, which is continuation of international application PCT/GB98/01197,filed Apr. 24, 1998, which itself is a continuation-in-part of U.S.provisional application No. 60/048,044, filed May 30, 1997.

This invention relates to diagnostic imaging techniques in which adisease state may be imaged using a targeted contrast agent and totargeted contrast agents suitable for use in such techniques. Moreparticularly the invention relates to the use of such contrast agents inwhich the targeting vector binds to receptors associated withangiogenesis. Such contrast agents may thus be used for diagnosis of forexample malignant diseases, heart diseases, inflammation-relateddiseases, rheumatoid arthritis and Kaposi's sarcoma. Moreover suchagents may be used in therapeutic treatment of these diseases.

New blood vessels can be formed by two different mechanisms:vasculogenesis or angiogenesis. Angiogenesis is the formation of newblood vessels by branching from existing vessels. The primary stimulusfor this process may be inadequate supply of nutrients and oxygen(hypoxia) to cells in a tissue. The cells may respond by secretingangiogenic factors, of which there are many; one example, which isfrequently referred to, is vascular endothelial growth factor (VEGF).These factors initiate the secretion of proteolytic enzymes which breakdown the proteins of the basement membrane, as well as inhibitors whichlimit the action of these potentially harmful enzymes. The otherprominent effect of angiogenic factors is to cause endothelial cells tomigrate and divide. Endothelial cells which are attached to the basementmembrane, which forms a continuous sheet around blood vessels on thecontralumenal side, do not undergo mitosis. The combined effect of lossof attachment and signals from the receptors for angiogenic factors isto cause the endothelial cells to move, multiply, and rearrangethemselves, and finally to synthesise a basement membrane around the newvessels.

Angiogenesis is prominent in the growth and remodeling of tissues,including wound healing and inflammatory processes. Tumors must initiateangiogenesis when they reach millimeter size in order to keep up theirrate of growth. As angiogenesis is accompanied by characteristic changesin the endothelial cells and their environment, this process is apromising target for therapeutic intervention. Inhibition ofangiogenesis is also considered to be a promising strategy for antitumortherapy. The transformations accompanying angiogenesis are also verypromising for diagnosis, an obvious example being malignant disease, butthe concept also shows great promise in inflammation and a variety ofinflammation-related diseases, including atherosclerosis, themacrophages of early atherosclerotic lesions being potential sources ofangiogenic factors. These factors are also involved inre-vascularisation of infarcted parts of the myocardium, which occurs ifa stenosis is released within a short time.

Angiogenesis involves receptors which are unique to endothelial cells.The surface of these cells is remodelled in preparation for migration,and cryptic structures are exposed where the basement membrane isdegraded, in addition to the variety of proteins which are involved ineffecting and controlling proteolysis. A number of knownreceptors/targets associated with angiogenesis are listed in Table 1below. In the case of tumors, the resulting network of blood vessels isusually disorganised, with the formation of sharp kinks and alsoarteriovenous shunts. Using the targeting principles described in thepresent disclosure, angiogenesis may be detected by the majority of theimaging modalities in use in medicine.

TABLE 1 Receptors/targets associated with angiogenesis Receptors/Targetsα₂-antiplasmin basement membrane components basic fibroblast growthfactor (bFGF) biglycan (dermatan sulfate proteoglycan) cartilage-derivedinhibitor [inhibitor] CD34 collagen type I, IV, VI, VIII decorin(dermatan sulfate proteoglycan) dermatan sulfate proteoglycans endoglinendosialin endothelin epidermal growth factor (heparin-binding) fibrinfibrinopeptide B fibroblast growth factor, FGF-3, basic fibronectinFlt-1/KDR, Flt-4 (VEGF receptor) FLT-1 (fms-like tyrosine kinase)(VEGF-A receptor) heparan sulfate hepatocyte growth factor hepatocytegrowth factor receptor (c-met) hyaluronan insulin-like growth factorinsulin-like growth factor/mannose-6-phosphate receptor integrins: β₃and β₅, integrin α_(v)β₃, integrin α₆β₁ (laminin receptor), integrin α₆,integrin β₁, integrin α₂β₁, integrin α₅ (subunit of the fibronectinreceptor), integrin α_(v)β₅, fibrin receptors interferon-α, β[inhibitors] interleukins: IL-8, IL-12 [inhibitor] Jagged gene product.laminin laminin fragments leukemia inhibitory factor Ly-6 (a lymphocyteactivation protein) matrix metalloprotease-2 metalloproteinasesmetalloproteinase inhibitors MHC class II Notch gene product placentalgrowth factor placental proliferin-related protein plasmiflogenplasminogen activator plasminogen activator inhibitor-1 plasminogenactivator receptor platelet-derived growth factor (e.g. type BB)platelet-derived endothelial cell growth factor platelet factor 4[inhibitor] pleiotropin proliferin, proliferin-related protein receptortyrosine kinases selectins: E-selectin SPARC stress proteins (molecularcharperones) (glucose regulated, heat shock families) syndecan tissueinhibitor of metalloproteinases (e.g. TIMP-2) thrombinthrombin-receptor-activating tetradecapeptide thrombospondin [inbibitor]TIE receptors (tyrosine kinases with Ig- and EGF-like domains) tissuefactor transforming growth factor-α, β tumor growth factor-α tumornecrosis factor urokinase-type plasminogen activator receptor Vascularendothelial growth factor-A Vascular endothelial growth factor-relatedprotein Vascular endothelial growth factor-A receptor vitronectin vonWillebrand factor note: many hormones, growth factors and othercompounds which bind to cell surface receptors may act as vectors bybinding to their receptors, or, when they are already bound to the cellsurface, they are targets for vectors that bind to them, for instanceantibodies.

As indicated above, many undesired conditions are associated withneovascularization or angiogenesis, the development or proliferation ofnew blood vessels. Examples of such conditions are listed in Table 2below

TABLE 2 Diseases and indications associated with angiogenesisDiseases/Indications arteriovenous malformations astrocytomasatherosclerosis breast cancers choriocarcinomas colorectal cancersgingivitis glioblastomas gliomas hemangiomas (childhood, capillary)hepatomas hyperplastic endometrium inflammation (e.g. chronic) ischemicmyocardium Kaposi sarcoma lung cancers macular degeneration melanomametastasis neuroblastomas occluding peripheral artery diseaseosteoarthritis ovarian cancers pancreatic cancers prostate cancerspsoriasis retinopathy (diabetic, proliferative) rheumatoid arthritisscleroderma seminomas skin cancers solid tumor formation ulcerativecolitis

The surface cells, endothelial cells, of such new blood vessels havegreater than normal concentrations of various surface or transmembranereceptors, such as for example receptor tyrosine kinases (RTK), and ithas been proposed to use radiolabelled or chromophore-labelledantibodies to such receptors, or similarly labelled analogues of naturalprotein ligands for such receptors, as a means of detecting centres ofangiogenesis (see for example WO95/26364 (Orion), WO96/30046 (Genentech)and WO95/12414 (Repligen)).

Peptidic ligands however have relatively few attachment sites fordetectable labels (reporters) and attachment of reporters at many siteson such peptidic ligands will affect the conformations which the ligandmay adopt. A further problem with peptides is that they are oftenunstable in vivo.

There is therefore still a need for effective targeted contrast agentswith affinities for receptors associated with angiogenesis.

The present invention addresses this need in two ways—firstly byproviding targeted contrast agents based on non-peptidic ligands(vectors)—and secondly by providing targeted contrast agents based onmacromolecular or particulate reporters providing a multiplicity ofdetectable labels (multireporters).

Thus viewed from one aspect the invention provides a composition ofmatter of formula IV-L-R  (I)wherein V is a vector moiety having affinity for an angiogenesis-relatedendothelial cell receptor, L is a linker moiety or a bond, and R is adetectable reporter moiety, preferably a gas-free detectable reportermoiety, e.g. detectable in an imaging procedure, such as in vivo imagingof the human or vascularized non-human animal body (e.g. mammalian,avian or reptilian body), characterised in that V is a non-peptidicorganic group, or V is peptidic and R is a macromolecular or particulatespecies providing a multiplicity of labels detectable in in vivoimaging.

Where R is a macromolecular or particulate species providing amultiplicity of labels, these may be labels which individually aredetctable (e.g. paramagnetic or radioactive species) or they mayinteract to produce a detectable material, e.g. by virtue of acooperative magnetic phenomenon. Examples of such multi-reportersinclude polychelates and polyionic species, and ferromagnetic,ferrimagnetic and superparamagnetic particles.

In many instances, the composition of matter of formula I will be acharacterisable compound. In others it may be a combination of compoundsbonded or otherwise associated, eg. conjugated, with each other. Forconvenience sake, the composition of matter will be referred tohereinafter as an agent.

By “gas” is meant a material or mixture of materials which is gaseous at37° C. By “gas-free” is meant a reporter which does not containsufficient gas to be detectable in ultrasonography in vivo. Contrastagents comprising gas-containing reporters are described in ourcopending International Patent Application No. PCT/GB97/02958 filed 28Oct. 1997.

Viewed from a further aspect the invention provides a pharmaceuticalcomposition comprising an effective amount (eg. an amount effective toenhance image contrast in in vivo imaging) of an agent of formula Itogether with at least one pharmaceutically effective carrier orexcipient.

Viewed from a still further aspect the invention provides the use of anagent of formula I for the manufacture of a contrast medium for use in amethod of diagnosis involving administration of said contrast medium toan animate subject and generation of an image of at least part of saidsubject.

Viewed from a still further aspect the invention provides a method ofgenerating an image of an animate human or non-human (preferablymammalian or avian) animal subject involving administering a contrastagent to said subject, eq. into the vascular system or the gi tract, andgenerating an image of at least a part of said subject to which saidcontrast agent has distributed, eg. by X-ray, MR, ultrasound,scintigraphy, PET, SPECT, electrical impedance, light or magnetometricimaging modalities, characterised in that as said contrast agent is usedan agent of formula I.

Viewed from a further aspect the invention provides a method ofmonitoring the effect of treatment of a human or non-human animalsubject with a drug to combat a condition associated with angiogenesis,e.g. a cytotoxic agent, said method involving administering to saidsubject an agent of formula I and detecting the uptake of said agent byendothelial cell receptors, in particular receptors in a region ofangiogenesis, said administration and detection optionally butpreferably being effected repeatedly, eg. before, during and aftertreatment with said drug.

Viewed from a yet further aspect the invention provides a process forthe preparation of an agent of formula I, said process comprisingconjugating (i) compound having binding affinity for an endothelial cellreceptor associated with angiogenesis to (ii) a compound detectable in adiagnostic imaging procedure or a chelant compound and if necessarymetallating chelant groups in the resultant conjugate with a metal iondetectable in a diagnostic imaging procedure.

The agents of formula I have three characteristic components: a vector(V); a linker (L); and a reporter (R). The vector must have the abilityto target the compound to a region of angiogenesis, the reporter must bedetectable in an in vivo diagnostic imaging procedure; and the linkermust couple vector to reporter, at least until the reporter has beendelivered to the region of angiogenesis and-preferably until the imagingprocedure has been completed.

Vectors

As the vector may be used any peptidic or, more preferably, non-peptidiccompound having affinity for receptors associated with angiogenesis.

Non-peptidic compounds are preferably used as peptidic vectors willgenerally have poor biological stability and may provoke undesiredresponses by the body.

Preferably the vector is a compound which does not elicit anyunacceptable biological response, especially one which does not actuallypromote angiogenesis.

Particularly preferably the vector is an angiogenesis inhibitor,especially preferably a non-peptidic angiogenesis inhibitor.

Examples of non-peptidic angiogenesis inhibitors are described inWO94/17084 (British Biotech), EP-A-618208 (Daiichi), WO94/13277 (ICRT),WO95/06473 (Nippon Kayaku), WO94/21612 (Otsuka), WO97/37655 (Merck),WO97/30035 (Zeneca), EP-A-678296 (Tsumura), WO94/18967 (Harvard),WO95/08327 (Dept. of Health and Human Services) (see also U.S. Pat. No.4,590,201 (Merck)) and EP-A-652000 (Eli Lilly).

Examples of peptidic angiogenesis inhibitors are described in WO94/02446(British Biotech), WO94/02447 (British Biotech), WO94/21625 (BritishBiotech), WO94/24140 (British Biotech), WO95/04033 (Celltech),EP-A-589719 (Eli Lilly), U.S. Pat. No. 5,399,667 (Frazier), EP-A-241830(The General Hospital Corporation) and WO97/38009 (Merck).

Particular angiogenesis inhibitors under development include thoselisted in Table 3 below:

TABLE 3 Angiogenesis Inhibitors Compound Target indications CompanyCommemts Tecogalan sodium Kaposi's sarcoma Daiichi sulfated poly- Solidtumors saccharide peptido- glycan complex AGM-1470 Kaposi's sarcomaTakeda/ Fumagillin analog Malignant tumors Abbott CM101 Cancer CarbomedPolysaccharide Metastasis exotoxin Mitoflaxone Solid tumors LiphaGM-1603 Glycomed Modified heparin rPF4 Kaposi's sarcoma ReplistatinRecombinant human Colon cancer Repligen platelet factor-4 Glioma Renalcell carcinoma Malignant melanoma MPF-4 Lilly Modified human plateletfactor-4 Recombinant EntreMed angiostatin Endostatin collagen fragmentThalidomide Brain, breast and EntreMed prostate cancer DC101 ImCloneMonoclonal Systems antibody OLX-514 Solid tumors Aronex SepsisRaloxifene Lilly hydrochloride Suramin sodium Metastatic Parke-Davishormone- refractory prostate carcinoma IL-12 Kidney cancer RocheMarimastat Pancreatic, lung British and brain cancer Biotech CAI Widerange of NCI Ca channel blocker cancersas well as the following peptidic and non-peptidic drug compounds:

Other known compounds capable of targeting regions of angiogenesis arelisted in Table 4 below:

TABLE 4 Vector molecules with known affinity for receptors associatedwith angiogenesis Vector Molecules angiopoietins angiostatin[plasminogen fragment] [inhibitor] angiotensin II α₂-antiplasmincombinatorial libraries, compounds from for instance compounds that bindto basement membrane after degradation β-Cyclodextrin tetradecasulfateendoglin endosialin endostatin [collagen fragment] epidermal growthfactor (heparin-binding) fibrin fibrinopeptide B fibroblast growthfactor, FGF-3, basic fibronectin fumagillin and analogs heparinhepatocyte growth factor hyaluronan insulin-like growth factorinterferon-α, β [inhibitors] interleukins: IL-8, IL-12 [inhibitor]laminin, laminin fragments leukemia inhibitory factor linomide matrixmetalloproteinase-2 metalloproteinases metalloproteinase inhibitorsmonoclonal antibodies for instance: to angiogenic factors or theirreceptors or to components of the fibrinolytic system peptides: forinstance, cyclic RGD_(D)FV placental growth factor placentalproliferin-related protein plasminogen plasminogen activator plasminogenactivator inhibitor-1 platelet activating factor antagonists[inhibitors] platelet-derived growth factor (e.g. type BB)platelet-derived growth factor receptors platelet-derived endothelialcell growth factor pleiotropin proliferin, proliferin-related proteinselectins: E-selectin SPARC snake venoms (RGD-containing) substance P (aneuropeptide: neurokinin) suramin tissue inhibitor of metalloproteinases(e.g. TIMP-2) thalidomide thrombin thrombin-receptor-activatingtetradecapeptide transforming growth factor-α, β transforming growthfactor receptor tumor growth factor-α tumor necrosis factor vitronectinnote: many hormones, growth factors and other compounds which bind tocell surface receptors may act as vectors by binding to their receptors,or, when they are already bound to the cell surface, they are targetsfor vectors that bind to them, for instance antibodies.

Similarly the compounds described in WO95/08327 may be used as vectors(see also Kohn et al. Proc. Nat. Acad Sci. USA 92: 1307-1311 (1995) andJ. Clin. Oncol. 15: 1985-1993 (1997)).

Particular examples of vector compounds described in some of the patentpublications mentioned above are as follows:

WO95/08327 (Dept. of Health and Human Services) describes angiogenesisinhibitor compounds of formula I and II:Y—(CH₂)_(p)—Ar¹—X—Ar²  (I)wherein

-   Ar¹ and Ar² are aromatic groups and may be different or the same;    and X is a linking group eg. O, S, SO₂, CO, CHCN, alkyl, alkoxy or    alkoxyalkyl, and

wherein

-   A is N or CH; R1 is H, —CONH, —CONHR⁵, COOH, —COOR⁵, SO₂NH₂; R2 is    H, NH₂, NHCOPh, —NHCOR⁵, —NHCHO, —NHR⁵, —N(R⁵)₂; and R⁵ is alkyl    with 1-6 carbons, e.g.

WO95/06473 (Nippon Kayaku Kabushiki Kasai) discloses antitumor andangiogenesis inhibitor compounds of formula 1 and 2:

wherein

-   X is O, COO; and M is a transition metal, and

wherein

-   X₁ and X₂ are substituted or unsubstituted phenyl or naphthyl    groups; Y₁ and Y₂ are halogen atoms, amino groups or mono- or    di-substituted amino groups; and Z is NHC₂H₄NH or a substituted or    unsubstituted aromatic diamine residue e.g.

WO95/04033 (Celltech Limited) discloses the following angiogenesisinhibitors:

-   R3 is H, halogen or CH₃, CF₃ or OCH₃; R2 is H or CH₃ e.g.    N⁴-hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-4(chlorophenyl-propyl)succinamide;    N⁴-hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-methylphenyl-propyl)succinamide;    N⁴-hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-methoxyphenyl-propyl)succinamide;    and    N⁴-hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-trifluoromethylphenyl-propyl)-succinamide.

EP 241830 (The General Hospital Corporation) discloses purification ofhepatoma-derived growth factor (HDGF), which is an endothelial mitogenand a potent angiogenic factor. The use of HDGF in controllingangiogenesis and detecting cancerous liver tumors by use of animmunodiagnostic assay is also disclosed. The HDGF peptide fragment hasan N-terminal amino acid sequence wherein the first 16 amino acids are:

-   leu-pro-ala-leu-pro-glu-asp-gly-gly-xx-gly-ala-phe-pro-pro-gly (SEQ    ID NO: 1)-   (xx=unidentified amino acid moiety)

An HDGF peptide fragment is also disclosed which has an N-terminal aminoacid extension sequence comprising:

-   (ala/ser)-(leu/arg)-pro-(ala/gly)-(leu/pro)-ala-gly-thr-met-ala-(ala)-gly-ser-(isoleu)-thr-thr-leu    (SEQ ID NO: 2)

EP 652000 (Eli Lilly and Company) discloses angiogenesis inhibitor andangiogenic disease inhibitor compounds having the formula:

wherein

-   R1 and R3 are H, Me, —C(O)(C₁-C₄ alkyl), —C(O)Ar; Ar is optionally    substituted phenyl; and R2 is pyrrolidino or piperidino, e.g.

EP 652000 [589719?] (Eli Lilly and Company) discloses modified plateletfactor-4 having the amino acid sequence:

MPF-4

-   NH₂-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln-Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser-COOH    (SEQ ID NO: 3)    CPF-4-   NH₂-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln-Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser-COOH    (SEQ ID NO: 4) disulfide bonded to a second protein having the amino    acid sequence    NH₂-Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-COOH.    (SEQ ID NO: 5) There are disulfide bridges between Cys-20 of MPF-4    and Cys-10 of the said second protein and between Cys-36 of MPF-4    and Cys-12 of the said second protein.

WO94/13277 (Imperical Cancer Research Technology Limited) discloses theuse of compounds of formula I:

wherein

-   R1 to R4 are each independently one or more of —X, N₃, —NO₂, halo,    trifluoromethyl, R⁵, OR⁵, —CH₂OR⁶, —OCOR⁵, —CH₂OCOR⁵, —NHCOR⁵,    —CH₂NHCOR⁵, —NR⁵R⁶, —CH₂NR⁵R⁶, —CH₂NO₂, CONR⁵R⁶, CH₂CONR⁵R⁶, —COOR⁵,    —CH₂COOR⁵, —CHO and CH₂CHO and —X is independently —SO₃R⁵,    —CH₂PO₃R⁵R⁶, —CH₂SO₃R⁵, —OSO₃R⁵, —CH₂OSO₃R⁵, —NHSO₃R⁵, —CH₂NHSO₃R⁵,    OPO₃R⁵R⁶, —CH₂OPO₃R⁵R⁶ and —PO₃R⁵R⁶ where R⁵ and R⁶ are chosen    independently from —H and lower alkyl and wherein A is a chemical    group comprising at least 5 and no more than 30 bonds directly    linking the naphthyl groups provided that (i) the compound is not    suramin and (ii) when A is not

wherein

-   m and n are independently 0, 1 or 2, then at least one of R1 to R4    is —OH or an acidic group; and of the pharmaceutically acceptable    salts, esters, salts of such esters or amides of such compounds.

Also described are compounds wherein the linkage of A to the naphthylring is via an amide or sulphonamide group. Furthermore A may in somecases be a group of formula II. A may also be selected from straightchain or branched alkyl groups, aryl groups, alkylaryl groups, aliphaticdicarboxylic acids, polyenes and derivatives thereof and polyols andderivatives thereof. Some further compounds are of formulae:

EP 678296 (Tsumura & Co.) discloses angiogenesis inhibitors of thegeneral formula:

WO94/18967 (President and Fellows of Harvard College) discloses a classof imidazoles that inhibit angiogenesis:

EP-A-618208 (Daiichi Pharmaceutical) discloses compounds of formula:

WO94/02446 (British Biotechnology Limited) discloses compounds offormula:

WO94/02447 (British Biotechnology Limited) discloses compounds offormula:

WO94/21625 (British Biotechnology Limited) discloses compounds offormula:

WO94/24149 (British Biotechnology Limited) discloses compounds offormula (I):

wherein

-   X is —CONHOH or COOH, principally characterised by the presence in    substituent R3 and/or R4 of a group of formula (II)

The contents of all the publications referred to herein is herebyincorporated by reference.

Particularly preferred vectors include amino acid derivatives such asdescribed in WO94/02446, hydroxamic acid derivatives such as describedin WO94/02447, thiazolopyrimidines such as described in EP-A-618208,triazoles such as described in WO95/08327, quinazolines such asdescribed in WO97/30035, isoindolones such as described in WO97/37655,integrin inhibitors, VEGF antagonists, bFGF antagonists, thrombospondinand thrombospondin fragments, CD36 and growth factors (e.g. VEGF, bFGF,etc).

CAM-D and other candidate identification and evaluation techniques asmentioned above can also be used to find or assess further candidatepeptidic and non-peptidic vectors.

Thus it is also possible to obtain molecules that bind specifically toangiogenesis associated receptors by direct screening of molecularlibraries. Screening of peptidic libraries may also be used to identifygenerally effective peptidic structures of which non-peptidic analogsmay be generated by conventional or combinatorial chemistry. Bindingmoieties identified in this way may be coupled to a linker molecule,constituting a general tool for attaching any vector molecule (ormolecules) to the reporter. Vector molecules may be generated fromcombinatorial libraries without necessarily knowing the exact moleculartarget, by functionally selecting (in vitro, ex vivo or in vivo) formolecules binding to the region/structure to be imaged.

As mentioned above, the agents of formula I comprise vector, linker andreporter moieties. A linker moiety may serve to link one vector to onereporter; alternatively it may link together more than vector and/ormore than one reporter. Likewise a reporter or a vector may be linked tomore than one linker. Use in this way of a plurality of reporters (eg.several linker-reporter moieties attached to one vector or severalreporters attached to one linker itself attached to one vector) mayenable the detectability of the contrast agent to be increased (eg. byincreasing its radioopacity, echogenicity or relaxivity) or may enableit to be detected in more than one imaging modality. Use in this way ofa plurality of vectors may increase the targeting efficiency of thecontrast agent or may make the contrast agent able to target more thanone site, eg. different receptors for an agent which has receptorheterogeneity. Thus for example the agent of formula I may includevector moieties with affinity sites other than angiogenesis associatedreceptors, eg. with affinities for cell surfaces on body duct wallsurfaces. Accordingly, the agent may include vectors such as antibodyfragments and oligopeptides, eg. containing RGD or analogous cellsurface binding peptide motifs (for example as described in EP-A-422937and EP-A-422938 (Merck)) or other vectors as described in GB 9700699.3.Such extra vectors may also be selected from any of the molecules thatnaturally concentrate in a selected target organ, tissue, cell or groupof cells, or other location in a mammalian body, in vivo. These caninclude amino acids, oligopeptides (e.g. hexapeptides), molecularrecognition units (MRU's), single chain antibodies (SCA's), proteins,non-peptide organic molecules, Fab fragments, and antibodies. Examplesof site-directed molecules include polysaccharides (e.g. CCK andhexapeptides), proteins (such as lectins, asialofetuin, polyclonal IgG,blood clotting proteins (e.g. hirudin), lipoproteins and glycoproteins),hormones, growth factors, clotting factors (such as PF4), polymerizedfibrin fragments (e.g., E₁), serum amyloid precursor (SAP) proteins, lowdensity lipoprotein (LDL) precursors, serum albumin, surface proteins ofintact red blood cells, receptor binding molecules such as estrogens,liver-specific proteins/polymers such as galactosyl-neoglycoalbumin(NGA) (see Vera et al. in Radiology 151: 191 (1984))N-(2-hydroxy-propyl)methacrylamide (HMPA) copolymers with varyingnumbers of bound galactosamines (see Duncan et al., Biochim. Biophys.Acta 880:62 (1986)), and allyl and 6-aminohexyl glycosides (see Wong etal., Carbo. Res. 170:27 (1987)), and fibrinogen. The site-directedprotein can also be an antibody. The choice of antibody, particularlythe antigen specificity of the antibody, will depend on the particularintended target site for the agent. Monoclonal antibodies are preferredover polyclonal antibodies. Preparation of antibodies that react with adesired antigen is well known. Antibody preparations are availablecommercially from a variety of sources. Fibrin fragment E1 can beprepared as described by Olexa et al. in J. Biol. Chem. 254:4925 (1979).Preparation of LDL precursors and SAP proteins is described by de Beeret al. in J. Immunol. Methods 50:17 (1982). The above described articlesare incorporated herein by reference in their entirety.

It is especially preferred that such extra vectors should bind so as toslow but not prevent the motion of the agent in the bloodstream and toanchor it in place when it is bound to a receptor site associated withangiogenesis.

Functional groups (e.g. amino groups, hydroxyl groups, carboxy groups,thiol groups, etc) on the vector compound may be used for binding of thevector to the linker moiety or directly to the reporter moiety, e.g.using conventional chemical coupling techniques.

Where the vector is a peptidic compound, the reporter is amultireporter, e.g. a metallated polychelant (preferably a dendrimericpolychelant), a magnetic (preferably superparamagnetic) particle, avesicle containing contrast effective particles or a solution ofcontrast effective molecule, a polyionic species (e.g. a polymercarrying a multiplicity of ionic groups, preferably anionic groups, e.g.a carboxylate, phosphate or sulphonate polymer).

Where the vector is non-peptidic, the reporter may be a multireporter oralternatively may comprise one or a small number (e.g. up to 10) ofdetectable labels, e.g. chelated paramagnetic metal ions, covalentlybound or chelated radioisotopes, and chromophores (or fluorophores,etc). Where the reporter is or comprises a covalently boundradionuclide, this is preferably an iodine radionuclide rather than atritium or ¹³C atom

Linker

A wide variety of linkers can be used, including biodegradable linkersand biopolymers.

The linker component of the contrast agent is at its simplest a bondbetween the vector and reporter moieties. More generally however thelinker will provide a mono- or multi-molecular skeleton covalently ornon-covalently linking one or more vectors to one or more reporters, eq.a linear, cyclic, branched or reticulate molecular skeleton, or amolecular aggregate, with in-built or pendant groups which bindcovalently or non-covalently, eq. coordinatively, with the vector andreporter moieties or which encapsulate, entrap or anchor such moieties.

Thus linking of a reporter unit to a desired vector may be achieved bycovalent or non-covalent means, usually involving interaction with oneor more functional groups located on the reporter and/or vector.Examples of chemically reactive functional groups which may be employedfor this purpose include amino, hydroxyl, sulfhydryl, carboxyl, andcarbonyl groups, as well as carbohydrate groups, vicinal diols,thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl andphenolic groups.

Covalent coupling of reporter and vector may therefore be effected usinglinking agents containing reactive moities capable of reaction with suchfunctional groups. Examples of reactive moieties capable of reactionwith sulfhydryl groups include α-haloacetyl compounds of the typeX—CH₂CO— (where X=Br, Cl or I), which show particular reactivity forsulfhydryl groups but which can also be used to modify imidazolyl,thioether, phenol and amino groups as described by Gurd, F. R. N. inMethods Enzymol. (1967) 11, 532. N-Maleimide derivatives are alsoconsidered selective towards sulfhydryl groups, but may additionally beuseful in coupling to amino groups under certain conditions. Reagentssuch as 2-iminothiolane, e.g. as described by Traut, R. et al. inBiochemistry (1973) 12, 3266, which introduce a thiol group throughconversion of an amino group, may be considered as sulfhydryl reagentsif linking occurs through the formation of disulphide bridges. Thusreagents which introduce reactive disulphide bonds into either thereporter or the vector may be useful, since linking may be brought aboutby disulphide exchange between the vector and reporter; examples of suchreagents include Ellman's reagent (DTNB), 4,4′-dithiodipyridine,methyl-3-nitro-2-pyridyl disulphide and methyl-2-pyridyl disulphide(described by Kimura, T. et al. in Analyt. Biochem. (1982) 122, 271).

Examples of reactive moieties capable of reaction with amino groupsinclude alkylating and acylating agents. Representative alkylatingagents include:

-   i) α-haloacetyl compounds, which show specificity towards amino    groups in the absence of reactive thiol groups and are of the type    X—CH₂CO— (where X=Cl, Br or I), e.g. as described by Wong, Y-H. H.    in Biochemistry (1979) 24, 5337;-   ii) N-maleimide derivatives, which may react with amino groups    either through a Michael type reaction or through acylation by    addition to the ring carbonyl group as described by Smyth, D. G. et    al. in J. Am. Chem. Soc. (1960) 82, 4600 and Biochem. J. (1964) 91,    589;-   iii) aryl halides such as reactive nitrohaloaromatic compounds;-   iv) alkyl halides as described by McKenzie, J. A. et al. in J.    Protein Chem. (1988) 7, 581;-   v) aldehydes and ketones capable of Schiff's base formation with    amino groups, the adducts formed usually being stabilised through    reduction to give a stable amine;-   vi) epoxide derivatives such as epichlorohydrin and    bisoxiranes,which may react with amino, sulfhydryl or phenolic    hydroxyl groups;-   vii) chlorine-containing derivatives of s-triazines, which are very    reactive towards nucleophiles such as amino, sufhydryl and hydroxy    groups;-   viii) aziridines based on s-triazine compounds dET_(A)iled above,    e.g. as described by Ross, W. C. J. in Adv. Cancer Res. (1954) 2, 1,    which react with nucleophiles such as amino groups by ring opening;-   ix) squaric acid diethyl esters as described by Tietze, L. F. in    Chem. Ber. (1991) 124, 1215; and-   x) α-haloalkyl ethers, which are more reactive alkylating agents    than normal alkyl halides because of the activation caused by the    ether oxygen atom, e.g. as described by Benneche, T. et al. in    Eur. J. Med. Chem. (1993) 28, 463.

Representative amino-reactive acylating agents include:

-   i) isocyanates and isothiocyanates, particularly aromatic    derivatives, which form stable urea and thiourea derivatives    respectively and have been used for protein crosslinking as    described by Schick, A. F. et al. in J. Biol. Chem. (1961) 236,    2477;-   ii) sulfonyl chlorides, which have been described by Herzig, D. J.    et al. in Biopolymers (1964) 2, 349 and which may be useful for the    introduction of a fluorescent reporter group into the linker;-   iii) Acid halides;-   iv) Active esters such as nitrophenylesters or N-hydroxysuccinimidyl    esters;-   v) acid anhydrides such as mixed, symmetrical or    N-carboxyanhydrides;-   vi) other useful reagents for amide bond formation as described by    Bodansky, M. et al. in ‘Principles of Peptide Synthesis’ (1984)    Springer-Verlag;-   vii) acylazides, e.g. wherein the azide group is generated from a    preformed hydrazide derivative using sodium nitrite, e.g. as    described by Wetz, K. et al. in Anal. Biochem. (1974) 58, 347;-   viii) azlactones attached to polymers such as bis-acrylamide, e.g.    as described by Rasmussen, J. K. in Reactive Polymers (1991) 16,    199; and-   ix) Imidoesters , which form stable amidines on reaction with amino    groups, e.g. as described by Hunter, M. J. and Ludwig, M. L. in J.    Am. Chem. Soc. (1962) 84, 3491.

Carbonyl groups such as aldehyde functions may be reacted with weakprotein bases at a pH such that nucleophilic protein side-chainfunctions are protonated. Weak bases include 1,2-aminothiols such asthose found in N-terminal cysteine residues, which selectively formstable 5-membered thiazolidine rings with aldehyde groups, e.g. asdescribed by Ratner, S. et al. in J. Am. Chem. Soc. (1937) 59, 200.Other weak bases such as phenyl hydrazones may be used, e.g. asdescribed by Heitzman, H. et al. in Proc. Natl. Acad. Sci. USA (1974)71, 3537.

Aldehydes and ketones may also be reacted with amines to form Schiff'sbases, which may advantageously be stabilised through reductiveamination. Alkoxylamino moieties readily react with ketones andaldehydes to produce stable alkoxamines, e.g. as described by Webb, R.et al. in Bioconjugate Chem. (1990) 1, 96.

Examples of reactive moieties capable of reaction with carboxyl groupsinclude diazo compounds such as diazoacetate esters and diazoacetamides,which react with high specificity to generate ester groups, e.g. asdescribed by Herriot R. M. in Adv. Protein Chem. (1947) 3, 169.Carboxylic acid modifying reagents such as carbodiimides, which reactthrough O-acylurea formation followed by amide bond formation, may alsousefully be employed; linking may be facilitated through addition of anamine or may result in direct vector-receptor coupling. Useful watersoluble carbodiimides include1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (CMC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), e.g. as describedby Zot, H. G. and Puett, D. in J. Biol. Chem. (1989) 264, 15552. Otheruseful carboxylic acid modifying reagents include isoxazoliumderivatives such as Woodwards reagent K; chloroformates such asp-nitrophenylchloroformate; carbonyldiimidazoles such as1,1′-carbonyldiimidazole; and N-carbalkoxydihydroquinolines such asN-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

Other potentially useful reactive moieties include vicinal diones suchas p-phenylenediglyoxal, which may be used to react with guanidinylgroups, e.g. as described by Wagner et al. in Nucleic acid Res. (1978)5, 4065; and diazonium salts, which may undergo electrophilicsubstitution reactions, e.g. as described by Ishizaka, K. and IshizakaT. in J. Immunol. (1960) 85, 163. Bis-diazonium compounds are readilyprepared by treatment of aryl diamines with sodium nitrite in acidicsolutions. It will be appreciated that functional groups in the reporterand/or vector may if desired be converted to other functional groupsprior to reaction, e.g. to confer additional reactivity or selectivity.Examples of methods useful for this purpose include conversion of aminesto carboxylic acids using reagents such as dicarboxylic anhydrides;conversion of amines to thiols using reagents such asN-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride,2-iminothiolane or thiol-containing succinimidyl derivatives; conversionof thiols to carboxylic acids using reagents such as α-haloacetates;conversion of thiols to amines using reagents such as ethylenimine or2-bromoethylamine; conversion of carboxylic acids to amines usingreagents such as carbodiimides followed by diamines; and conversion ofalcohols to thiols using reagents such as tosyl chloride followed bytransesterification with thioacetate and hydrolysis to the thiol withsodium acetate.

Vector-reporter coupling may also be effected using enzymes aszero-length crosslinking agents; thus, for example, transglutaminase,peroxidase and xanthine oxidase have been used to produce crosslinkedproducts. Reverse proteolysis may also be used for crosslinking throughamide bond formation.

Non-covalent vector-reporter coupling may, for example, be effected byelectrostatic charge interactions, through chelation in the form ofstable metal complexes or through high affinity binding interaction.

A vector which is coupled to a peptide, lipo-oligosaccharide orlipopeptide linker which contains a element capable of mediatingmembrane insertion may also be useful. One example is described byLeenhouts, J. M. et al. in Febs Letters (1995) 370(3), 189-192.

Coupling may also be effected using avidin or streptavidin, which havefour high affinity binding sites for biotin. Avidin may therefore beused to conjugate vector to reporter if both vector and reporter arebiotinylated. Examples are described by Bayer, E. A. and Wilchek, M. inMethods Biochem. Anal. (1980) 26, 1. This method may also be extended toinclude linking of reporter to reporter, a process which may encourageassociation of the agent and consequent potentially increased efficacy.Alternatively, avidin or streptavidin may be attached directly to thesurface of reporter particles.

Non-covalent coupling may also utilise the bifunctional nature ofbispecific immunoglobulins. These molecules can specifically bind twoantigens, thus linking them. For example, either bispecific IgG orchemically engineered bispecific F(ab)′₂ fragments may be used aslinking agents. Heterobifunctional bispecific antibodies have also beenreported for linking two different antigens, e.g. as described by Bode,C. et al. in J. Biol. Chem. (1989) 264, 944 and by Staerz, U. D. et al.in Proc. Natl. Acad. Sci. USA (1986) 83, 1453. Similarly, any reporterand/or vector containing two or more antigenic determinants (e.g. asdescribed by Chen, Aa et al. in Am. J. Pathol. (1988) 130, 216) may becrosslinked by antibody molecules and lead to formation of cross-linkedassemblies of agents of formula I of potentially increased efficacy.

So-called zero-length linking agents, which induce direct covalentjoining of two reactive chemical groups without introducing additionallinking material (e.g. as in amide bond formation induced usingcarbodiimides or enzymatically) may, if desired, be used in accordancewith the invention, as may agents such as biotin/avidin systems whichinduce non-covalent reporter-vector linking and agents which induceelectrostatic interactions.

Most commonly, however, the linking agent will comprise two or morereactive moieties, e.g. as described above, connected by a spacerelement. The presence of such a spacer permits bifunctional linkers toreact with specific functional groups within a molecule or between twodifferent molecules, resulting in a bond between these two componentsand introducing extrinsic linker-derived material into thereporter-vector conjugate. The reactive moieties in a linking agent maybe the same (homobifunctional agents) or different (heterobifunctionalagents or, where several dissimilar reactive moieties are present,heteromultifunctional agents), providing a diversity of potentialreagents that may bring about covalent bonding between any chemicalspecies, either intramolecularly or intermolecularly.

The nature of extrinsic material introduced by the linking agent mayhave a critical bearing on the targeting ability and general stabilityof the ultimate product. Thus it may be desirable to introduce labilelinkages, e.g. containing spacer arms which are biodegradable orchemically sensitive or which incorporate enzymatic cleavage sites.Alternatively the spacer may include polymeric components, e.g. to actas surfactants and enhance the stability of the agent. The spacer mayalso contain reactive moieties, e.g. as described above to enhancesurface crosslinking.

Spacer elements may typically consist of aliphatic chains whicheffectively separate the reactive moieties of the linker by distances ofbetween 5 and 30 Å. They may also comprise macromolecular structuressuch as poly(ethylene glycols). Such polymeric structures, hereinafterreferred to as PEGs, are simple, neutral polyethers which have beengiven much attention in biotechnical and biomedical applications (seee.g. Milton Harris, J. (ed) “Poly(ethylene glycol) chemistry,biotechnical and biomedical applications” Plenum Press, New York, 1992).PEGs are soluble in most solvents, including water, and are highlyhydrated in aqueous environments, with two or three water moleculesbound to each ethylene glycol segment; this has the effect of preventingadsorption either of other polymers or of proteins onto PEG-modifiedsurfaces. PEGs are known to be nontoxic and not to harm active-proteinsor cells, whilst covalently linked PEGs are known to be non-immunogenicand non-antigenic. Furthermore, PEGs may readily be modified and boundto other molecules with only little effect on their chemistry. Theiradvantageous solubility and biological properties are apparent from themany possible uses of PEGs and copolymers thereof, including blockcopolymers such as PEG-polyurethanes and PEG-polypropylenes.

Appropriate molecular weights for PEG spacers used in accordance withthe invention may, for example, be between 120 Daltons and 20 kDaltons.

The major mechanism for uptake of particles by the cells of thereticuloendothelial system (RES) is opsonisation by plasma proteins inblood; these mark foreign particles which are then taken up by the RES.The biological properties of PEG spacer elements used in accordance withthe invention may serve to increase the circulation time of the agent ina similar manner to that observed for PEGylated liposomes (see e.g.Klibanov, A. L. et al. in FEBS Letters (1990) 268, 235-237 and Blume, G.and Cevc, G. in Biochim. Biophys. Acta (1990) 1029, 91-97). Increasedcoupling efficiency to areas of interest may also be achieved usingantibodies bound to the terminii of PEG spacers (see e.g. Maruyama, K.et al. in Biochim. Biophys. Acta (1995) 1234, 74-80 and Hansen, C. B. etal. in Biochim. Biophys. Acta (1995) 1239, 133-144).

Other representative spacer elements include structural-typepolysaccharides such as polygalacturonic acid, glycosaminoglycans,heparinoids, cellulose and marine polysaccharides such as alginates,chitosans and carrageenans; storage-type polysaccharides such as starch,glycogen, dextran and aminodextrans; polyamino acids and methyl andethyl esters thereof, as in homo- and co-polymers of lysine, glutamicacid and aspartic acid; and polypeptides, oligosaccharides andoligonucleotides, which may or may not contain enzyme cleavage sites.

In general, spacer elements may contain cleavable groups such as vicinalglycol, azo, sulfone, ester, thioester or disulphide groups. Spacerscontaining biodegradable methylene diester or diamide groups of formula-(Z)_(m).Y.X.C(R¹R²).X.Y.(Z)_(n)-[where X and Z are selected from —O—, —S—, and —NR— (where R is hydrogenor an organic group); each Y is a carbonyl, thiocarbonyl, sulphonyl,phosphoryl or similar acid-forming group: m and n are each zero or 1;and R¹ and R² are each hydrogen, an organic group or a group

—X.Y.(Z)_(m)-, or together form a divalent organic group] may also beuseful; as discussed in, for example, WO-A-9217436 such groups arereadily biodegraded in the presence of esterases, e.g. in vivo, but arestable in the absence of such enzymes. They may therefore advantageouslybe linked to therapeutic agents to permit slow release thereof.

Poly[N-(2-hydroxyethyl)methacrylamides] are potentially useful spacermaterials by virtue of their low degree of interaction with cells andtissues (see e.g. Volfová, I., Ríhová, B. and V. R. and Vetvicka, P. inJ. Bioact. Comp. Polymers (1992) 7, 175-190). Work on a similar polymerconsisting mainly of the closely related 2-hydroxypropyl derivativeshowed that it was endocytosed by the mononuclear phagocyte system onlyto a rather low extent (see Goddard, P., Williamson, I., Bron, J.,Hutchkinson, L. E., Nicholls, J. and Petrak, K. in J. Bioct. Compat.Polym. (1991) 6, 4-24.).

Other potentially useful poymeric spacer materials include:

-   i) copolymers of methyl methacrylate with methacrylic acid; these    may be erodible (see Lee, P. I. in Pharm. Res. (1993) 10, 980) and    the carboxylate substituents may cause a higher degree of swelling    than with neutral polymers;-   ii) block copolymers of polymethacrylates with biodegradable    polyesters (see e.g. San Roman, J. and Guillen-Garcia, P. in    Biomaterials (1991) 12, 236-241);-   iii) cyanoacrylates, i.e. polymers of esters of 2-cyanoacrylic    acid—these are biodegradable and have been used in the form of    nanoparticles for selective drug delivery (see Forestier, F.,    Gerrier, P., Chaumard, C., Quero, A. M., Couvreur, P. and    Labarre, C. in J. Antimicrob. Chemoter. (1992) 30, 173-179);-   iv) polyvinyl alcohols, which are water-soluble and generally    regarded as biocompatible (see e.g. Langer, R. in J. Control.    Release (1991) 16, 53-60);-   v) copolymers of vinyl methyl ether with maleic anhydride, which    have been stated to be bioerodible (see Finne, U., Hannus, M. and    Urtti, A. in Int. J. Pharm. (1992) 78. 237-241);-   vi) polyvinylpyrrolidones, e.g. with molecular weight less than    about 25,000, which are rapidly filtered by the kidneys (see Hespe,    W., Meier, A. M. and Blankwater, Y. M. in Arzeim.-Forsch./Drug    Res. (1977) 27, 1158-1162);-   vii) polymers and copolymers of short-chain aliphatic hydroxyacids    such as glycolic, lactic, butyric, valeric and caproic acids (see    e.g. Carli, F. in Chim. Ind. (Milan) (1993) 75, 494-9), including    copolymers which incorporate aromatic hydroxyacids in order to    increase their degradation rate (see Imasaki, K., Yoshida, M.,    Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H. and    Nagai. T. in Int. J. Pharm. (1992) 81, 31-38);-   viii) polyesters consisting of alternating units of ethylene glycol    and terephthalic acid, e.g. Dacron^(R), which are non-degradable but    highly biocompatible;-   ix) block copolymers comprising biodegradable segments of aliphatic    hydroxyacid polymers (see e.g. Younes, H., Nataf, P. R., Cohn, D.,    Appelbaum, Y. J., Pizov, G. and Uretzky, G. in Biomater. Artif.    Cells Artif. Organs (1988) 16, 705-719), for instance in conjunction    with polyurethanes (see Kobayashi, H., Hyon, S. H. and Ikada, Y. in    “Water-curable and biodegradable prepolymers”—J. Biomed. Mater.    Res. (1991) 25, 1481-1494);-   x) polyurethanes, which are known to be well-tolerated in implants,    and which may be combined with flexible “soft” segments, e.g.    comprising poly(tetra methylene glycol), poly(propylene glycol) or    poly(ethylene glycol)) and aromatic “hard” segments, e.g. comprising    4,4′-methylenebis(phenylene isocyanate) (see e.g. Ratner, B. D.,    Johnston, A. B. and Lenk, T. J. in J. Biomed. Mater. Res: Applied    Biomaterials (1987) 21, 59-90; Sa Da Costa, V. et al. in J. Coll.    Interface Sci. (1981) 80, 445-452 and Affrossman, S. et al. in    Clinical Materials (1991) 8, 25-31);-   xi) poly(1,4-dioxan-2-ones), which may be regarded as biodegradable    esters in view of their hydrolysable ester linkages (see e.g.    Song, C. X., Cui, X. M. and Schindler, A. in Med. Biol. Eng.    Comput. (1993) 31, S147-150), and which may include glycolide units    to improve their absorbability (see Bezwada, R. S., Shalaby, S. W.    and Newman, H. D. J. in Agricultural and synthetic polymers:    Biodegradability and utilization (1990) (ed Glass, J. E. and Swift,    G.), 167-174—ACS symposium Series, #433, Washington D.C.,    U.S.A.—American Chemical Society);-   xii) polyanhydrides such as copolymers of sebacic acid (octanedioic    acid) with bis(4-carboxy-phenoxy)propane, which have been shown in    rabbit studies (see Brem, H., Kader, A., Epstein, J. I., Tamargo, R.    J., Domb, A., Langer, R. and Leong, K. W. in Sel. Cancer    Ther. (1989) 5, 55-65) and rat studies (see Tamargo, R. J.,    Epstein, J. I., Reinhard, C. S., Chasin, M. and Brem, H. in J.    Biomed. Mater. Res. (1989) 23, 253-266) to be useful for controlled    release of drugs in the brain without evident toxic effects;-   xiii) biodegradable polymers containing ortho-ester groups, which    have been employed for controlled release in vivo (see Maa, Y. F.    and Heller, J. in J. Control. Release (1990) 14, 21-28); and-   xiv) polyphosphazenes, which are inorganic polymers consisting of    alternate phosphorus and nitrogen atoms (see Crommen, J. H.,    Vandorpe, J. and Schacht, E. H. in J. Control. Release (1993) 24,    167-180).

The following tables list linking agents which may be useful intargetable agents in accordance with the invention.

Heterobifunctional Linking Agents

Linking agent Reactivity 1 Reactivity 2 Comments ABH carbohydratephotoreactive ANB-NOS —NH₂ photoreactive APDP (1) —SH photoreactiveiodinable disulphide linker APG —NH₂ photoreactive reacts selectivelywith Arg at pH 7-8 ASIB (1) —SH photoreactive iodinable ASBA (1) —COOHphotoreactive iodinable EDC —NH₂ —COOH zero-length linker GMBS —NH₂ —SHsulfo-GMBS —NH₂ —SH water-soluble HSAB —NH₂ photoreactive sulfo-HSAB—NH₂ photoreactive water-soluble MBS —NH₂ —SH sulfo-MBS —NH₂ —SHwater-soluble M₂C₂H carbohydrate —SH MPBH carbohydrate —SH NHS-ASA (1)—NH₂ photoreactive iodinable sulfo-NHS- —NH₂ photoreactivewater-soluble, ASA (1) iodinable sulfo-NHS-LC- —NH₂ photoreactivewater-soluble, ASA (1) iodinable PDPH carbohydrate —SH disulphide linkerPNP-DTP —NH₂ photoreactive SADP —NH₂ photoreactive disulphide linkersulfo-SADP —NH₂ photoreactive water-soluble disulphide linker SAED —NH₂photoreactive disulphide linker SAND —NH₂ photoreactive water-solubledisulphide linker SANPAH —NH₂ photoreactive sulfo-SANPAH —NH₂photoreactive water-soluble SASD (1) —NH₂ photoreactive water-solubleiodinable disulphide linker SIAB —NH₂ —SH sulfo-SIAB —NH₂ —SHwater-soluble SMCC —NH₂ —SH sulfo-SMCC —NH₂ —SH water-soluble SMPB —NH₂—SH sulfo-SMPB —NH₂ —SH water-soluble SMPT —NH₂ —SH sulfo-LC-SMPT —NH₂—SH water-soluble SPDP —NH₂ —SH sulfo-SPDP —NH₂ —SH water-solublesulfo-LC-SPDP —NH₂ —SH water-soluble sulfo-SAMCA (2) —NH₂ photoreactivesulfo-SAPB —NH₂ photoreactive water-soluble Notes: (1) = iodinable; (2)= fluorescentHomobifunctional Linking Agents

Linking agent Reactivity Comments BS —NH₂ BMH —SH BASED (1)photoreactive iodinable disulphide linker BSCOES —NH₂ sulfo-BSCOES —NH₂water-soluble DFDNB —NH₂ DMA —NH₂ DMP —NH₂ DMS —NH₂ DPDPB —SH disulphidelinker DSG —NH₂ DSP —NH₂ disulphide linker DSS —NH₂ DST —NH₂ sulfo-DST—NH₂ water-soluble DTBP —NH₂ disulphide linker DTSSP —NH₂ disulphidelinker EGS —NH₂ sulfo-EGS —NH₂ water-soluble SPBP —NH₂Biotinylation Agents

Agent Reactivity Comments biotin-BMCC —SH biotin-DPPE* preparation ofbiotinylated liposomes biotin-LC-DPPE* preparation of biotinylatedliposomes biotin-HPDP —SH disulphide linker biotin-hydrazidecarbohydrate biotin-LC-hydrazide carbohydrate iodoacetyl-LC-biotin —NH₂NHS-iminobiotin —NH₂ reduced affinity for avidin NHS-SS-biotin —NH₂disulphide linker photoactivatable biotin nucleic acids sulfo-NHS-biotin—NH₂ water-soluble sulfo-NHS-LC-biotin —NH₂ Notes: DPPE =dipalmitoylphosphatidylethanolamine; LC = long chainAgents for Protein Modification

Agent Reactivity Function Ellman's reagent —SHquantifies/detects/protects DTT —S•S— reduction 2-mercaptoethanol —S•S—reduction 2-mercaptylamine —S•S— reduction Traut's reagent —NH₂introduces —SH SATA —NH₂ introduces protected —SH AMCA-NHS —NH₂fluorescent labelling AMCA-hydrazide carbohydrate fluorescent labellingAMCA-HPDP —S•S— fluorescent labelling SBF-chloride —S•S— fluorescentdetection of —SH N-ethylmaleimide —S•S— blocks —SH NHS-acetate —NH₂blocks and acetylates —NH₂ citraconic —NH₂ reversibly blocks andanhydride introduces negative charges DTPA —NH₂ introduces chelatorBNPS-skatole tryptophan cleaves tryptophan residue Bolton-Hunter —NH₂introduces iodinable group para-iodophenylalanine

In addition to the already contemplated straight chain and branchedPEG-like linkers (e.g polyethylene glycols and other containing 2 to 100recurring units of ethylene oxide), linkers in the VLR system can beindependently a chemical bond or the residue of a linking group. Thephrase “residue of a linking group” as used herein refers to a moietythat remains, results, or is derived from the reaction of a vectorreactive group with a reactive site on a vector. The phrase “vectorreactive group” as used herein refers to any group which can react withfunctional groups typically found on vectors, the derivatization ofwhich only minimally effects the ability of the vector to bind to itsreceptor. However, it is specifically contemplated that such vectorreactive groups can also react with functional groups typically found onrelevant protein molecules. Thus, in one aspect the linkers useful inthe practice of this invention derive from those groups which can reactwith any relevant molecule which comprises a vector as described abovecontaining a reactive group, whether or not such relevant molecule is aprotein, to form a linking group.

Preferred linking groups are derived from vector reactive groupsselected from but not limited to:—

a group that will react directly with carboxy, aldehyde, amine (NHR),alcohols, sulfhydryl groups, activated methylenes and the like, on thevector, for example, active halogen containing groups including, forexample, chloromethylphenyl groups and chloroacetyl [ClCH₂C(═O)—]groups, activated 2-(leaving group substituted)-ethylsulfonyl andethylcarbonyl groups such as 2-chloroethylsulfonyl and2-chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl; epoxy; isocyanato;isothiocyanato; aldehyde; aziridine; succinimidoxycarbonyl; activatedacyl groups such as carboxylic acid halides; mixed anhydrides and thelike.

A group that can react readily with modified vector molecules containinga vector reactive group, i.e., vectors containing a reactive groupmodified to contain reactive groups such as those mentioned in thetables above, for example, by oxidation of the vector to an aldehyde ora carboxylic acid, in which case the “linking group” can be derived fromreactive groups selected from amino, alkylamino, arylamino, hydrazino,alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido,thiosemicarbazido, sulfhydryl, sulfhydrylalkyl, sulfhydrylaryl, hydroxy,carboxy, carboxyalkyl and carboxyaryl. The alkyl portions of saidlinking groups can contain from 1 to about 20 carbon atoms. The arylportions of said linking groups can contain from about 6 to about 20carbon atoms; and

a group that can be linked to the vector containing a reactive group, orto the modified vector as noted above by use of a crosslinking agent.The residues of certain useful crosslinking agents, such as, forexample, homobifunctional and heterobifunctional gelatin hardeners,bisepoxides, and bisisocyanates can become a part of a linking groupduring the crosslinking reaction. Other useful crosslinking agents,however, can facilitate the crosslinking, for example, as consumablecatalysts, and are not present in the final conjugate. Examples of suchcrosslinking agents are carbodiimide and carbamoylonium crosslinkingagents as disclosed in U.S. Pat. No. 4,421,847 and the ethers of U.S.Pat. No. 4,877,724. With these crosslinking agents, one of the reactantssuch as the vector must have a carboxyl group and the other such as along chain spacer must have a reactive amine, alcohol, or sulfhydrylgroup. In amide bond formation, the crosslinking agent first reactsselectively with the carboxyl group, then is split out during reactionof the thus “activated” carboxyl group with an amine to form an amidelinkage between thus covalently bonding the two moieties. An advantageof this approach is that crosslinking of like molecules, e.g., vector tovector is avoided, whereas the reaction of, for example,homo-bifunctional crosslinking agents is nonselective and unwantedcrosslinked molecules are obtained.

Preferred useful linking groups are derived from variousheterobifunctional cross-linking reagents such as those listed in thePierce Chemical Company Immunotechnology Catalog—Protein ModificationSection, (1995 and 1996). Useful non-limiting examples of such reagentsinclude:

-   Sulfo-SMCC Sulfosuccinimidyl    4-(N-maleimidomethyl)cyclohexane-1-carboxylate.-   Sulfo-SIAB Sulfosuccinimidyl (4-iodoacetyl)aminobenzoate.-   Sulfo-SMPB Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate.-   2-IT 2-Iminothiolane.-   SATA N-Succinimidyl S-acetylthioacetate.

In addition to the foregoing description, the linking groups, in wholeor in part, can also be comprised of and derived from complementarysequences of nucleotides and residues of nucleotides, both naturallyoccurring and modified, preferably non-self-associating oligonucleotidesequences. Particularly useful, non-limiting reagents for incorporationof modified nucleotide moieties containing reactive functional groups,such as amine and sulfhydryl groups, into an oligonucleotide sequenceare commercially available from, for example, Clontech Laboratories Inc.(Palo Alto Calif.) and include Uni-Link AminoModifier (Catalog # 5190),Biotin-ON phosphoramidite (Catalog # 5191), N-MNT-C6-AminoModifier(Catalog # 5202), AminoModifier II (Catalog # 5203), DMT-C6-3′Amine-ON(Catalog # 5222), C6-ThiolModifier (Catalog # 5211), and the like. Inone aspect, linking groups of this invention are derived from thereaction of a reactive functional group such as an amine or sulfhydrylgroup as are available in the above Clontech reagents, one or more ofwhich has been incorporated into an oligonucleotide sequence, with, forexample, one or more of the previously described vector reactive groupssuch as a heterobifunctional group on the vector.

By attaching two complementary oligonucleotide sequences one to thevector and the other to the reporter the resulting double-strandedhybridized oligonucleotide then comprises the linking group between thevector and reporter.

Other polymer systems that serve as linkers include:—

-   Poly(L or D or DL-amino acids)=proteins and peptides; naturally    occuring or synthetic-   Pseudo Poly(amino acids)=(amino acids linked by non-amide bonds)-   Poly (L or D or DL-lactide) and the co-polymers e.g-   Poly (L-lactide/DL-lactide)Poly (glycolide)-   L-lactide/glycolide co-polymers-   Poly-caprolactone and its co-polymers-   Polyanhydrides-   Poly (ortho esters)-   Polyphosphazenes-   Long-chain straight or branched lipids (& phospholipids)-   Sugars and carbohydrates-   Oligonucleotides (see above)-   as well as mixtures of the above.    Linking agents used in accordance with the invention will in general    bring about linking of vector to reporter or reporter to reporter    with some degree of specificity, and may also be used to attach one    or more therapeutically active agents.

The present invention accordingly provides a tool for therapeutic drugdelivery in combination with vector-mediated direction of the product tothe desired site. By “therapeutic” or “drug” is meant an agent having abeneficial effect on a specific disease in a living human or non-humananimal.

Therapeutic compounds used in accordance with the present invention maybe encapsulated in the interior of a molecular aggregate or particulatelinker or attached to or incorporated in the encapsulating walls of avesicular linker. Thus, the therapeutic compound may be linked to a partof the surface, for example through covalent or ionic bonds, or may bephysically mixed into an encapsulating material, particularly if thedrug has similar polarity or solubility to the material, so as toprevent it from leaking out of the product before it is intended to actin the body. The release of the drug may be initiated merely by wettingcontact with blood following administration or as a consequence of otherinternal or external influences, e.g. dissolution processes catalyzed byenzymes or the use of magnetic heating where the reporter is a magneticparticle.

The therapeutic substance may be covalently linked to the encapsulatingmembrane surface of a vascular linker using a suitable linking acent,e.g. as described herein. Thus, for example, one may initially prepare aphospholipid derivative to which the drug is bonded through abiodegradable bond or linker, and then incorporate this derivative intothe material used to prepare the vesicle membrane, as described above.

Alternatively, the agent may initially be prepared without thetherapeutic, which may then be coupled to or coated onto particulate(eg. vesicular) agents prior to use. Thus, for example, a therapeuticcould be added to a suspension of liposomes in aqueous media and shakenin order to attach or adhere the therapeutic to the liposomes.

The therapeutic may for example be a drug or prodrug known for use incombatting angiogenesis or tumors.

By targeting an agent according to the invention containing aprodrug-activating enzyme to areas of pathology one may image targetingof the enzyme, making it possible to visualise when the agent istargeted properly and when the agent has disappeared from non-targetareas. In this way one can determine the optimal time for injection ofprodrug into individual patients.

Another alternative is to incorporate a prodrug, a prodrug-activatingenzyme and a vector in the same particulate linker reporter in such away that the prodrug will only be activated after some externalstimulus. Such a stimulus may, for example, be light stimulation of achromophoric reporter, or magnetic heating of a superparamagneticreporter after the desired targeting has been achieved.

So-called prodrugs may also be used in agents according to theinvention. Thus drugs may be derivatised to alter their physicochemicalproperties and to adapt agent of the invention; such derivatised drugsmay be regarded as prodrugs and are usually inactive until cleavage ofthe derivatising group regenerates the active form of the drug.

Therapeutics may easily be delivered in accordance with the invention tosites of angiogenesis.

By way of example, where the reporter is a chelated metal species (eg. aparamagnetic metal ion or a metal radionuclide), the linker may comprisea chain attached to a metal chelating group, a polymeric chain with aplurality of metal chelating groups pendant from the molecular backboneor incorporated in the molecular backbone, a branched polymer with metalchelating groups at branch termini (eg. a dendrimeric polychelant), etc.What is required of the linker is simply that it bind the vector andreporter moieties together for an adequate period. By adequate period ismeant a period sufficient for the contrast agent to exert its desiredeffects, eg. to enhance contrast in vivo during a diagnostic imagingprocedure.

Thus, in certain circumstances, it may be desirable that the linkerbiodegrade after administration. By selecting an appropriatelybiodegradable linker it is possible to modify the biodistribution andbioelimination patterns for the vector and/or reporter. Where vectorand/or reporter are biologically active or are capable of exertingundesired effects if retained after the imaging procedure is over, itmay be desirable to design in linker biodegradability which ensuresappropriate bioelimination or metabolic breakdown of the vector and/orreporter moieties. Thus a linker may contain a biodegradable functionwhich on breakdown yields breakdown products with modifiedbiodistribution patterns which result from the release of the reporterfrom the vector or from fragmentation of a macromolecular structure. Byway of example for linkers which carry chelated metal ion reporters itis possible to have the linker incorporate a biodegradable functionwhich on breakdown releases an excretable chelate compound containingthe reporter.

Accordingly, biodegradable functions may if desired be incorporatedwithin the linker structure, preferably at sites which are (a) branchingsites, (b) at or near attachment sites for vectors or reporters, or (c)such that biodegradation yields physiologically tolerable or rapidlyexcretable fragments.

Examples of suitable biodegradable functions include ester, amide,double ester, phosphoester, ether, thioether, guanidyl, acetal and ketalfunctions.

As discussed above, the linker group may if desired have built into itsmolecular backbone groups which affect the biodistribution of thecontrast agent or which ensure appropriate spatial conformation for thecontrast agent, eg. to allow water access to chelated paramagnetic metalion reporters. By way of example the linker backbone may consist in partor essentially totally of one or more polyalkylene oxide chains.

Thus the linker may be viewed as being a composite of optionallybiodegradable vector binding (V_(b)) and reporter binding (R_(b)) groupsjoined via linker backbone (L_(b)) groups, which linker backbone groupsmay carry linker side chain (L_(sc)) groups to modify biodistributionetc. and may themselves incorporate biodegradable functions. The R_(b)and V_(b) binding groups may be pendant from the linker backbone or maybe at linker backbone termini, for example with one R_(b) or V_(b) groupat one L_(b) terminus, with R_(b) or V_(b) groups linking together twoL_(b) termini or with one L_(b) terminus carrying two or more R_(b) orV_(b) groups. The L_(b) and L_(sc) groups will conveniently beoligomeric or polymeric structures (eg. polyesters, polyamides,polyethers, polyamines, oligopeptides, polypeptides, oligo andpolysaccharides, oligonucleotides, etc.), preferably structures havingat least in part a hydrophilic or lipophilic nature, eg. hydrophilic,amphiphilic or lipophilic structures.

The linker may be low, medium or high molecular weight, eq. up to 2MD.Generally higher molecular weight linkers will be preferred if they areto be loaded with a multiplicity of vectors or reporters or if it isnecessary to space vector and reporter apart, or if the linker is itselfto serve a role in the modification of biodistribution. In generalhowever linkers will be from 100 to 100 000 D, especially 120 D to 20 kDin molecular weight.

Conjugation of linker to vector and linker to reporter may be by anyappropriate chemical conjugation technique, eg. covalent bonding (forexample ester or amide formation), metal chelation or other metalcoordinative or ionic bonding, again as described above.

Examples of suitable linker systems include the magnifier polychelantstructures of U.S. Pat. No. 5,364,613 and PCT/EP90/00565, polyaminoacids(eg. polylysine), functionalised PEG, polysaccharides,glycosaminoglycans, dendritic polymers such as described in WO93/06868and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990),PEG-chelant polymers such as described in W94/08629, WO94/09056 andWO96/26754, etc.

Where the reporter is a chelated metal ion, the linker group willgenerally incorporate the chelant moiety. Alternatively, the chelatedmetal may be carried on or in a particulate reporter. In either case,conventional metal chelating groups such as are well known in the fieldsof radiopharmaceuticals and MRI contrast media may be used, eg. linear,cyclic and branched polyamino-polycarboxylic acids and phosphorusoxyacid equivalents, and other sulphur and/or nitrogen ligands known inthe art, eg. DTPA, DTPA-BMA, EDTA, D03A, TMT (see for example U.S. Pat.No. 5,367,080), BAT and analogs (see for example Ohmono et al., J. Med.Chem. 35: 157-162 (1992) and Kung et al. J. Nucl. Med. 25: 326-332(1984)), the N₂S₂ chelant ECD of Neurolite, MAG (see Jurisson et al.Chem. Rev. 93: 1137-1156 (1993)), HIDA, DOXA(1-oxa-4,7,10-triazacyclododecanetriacetic acid), NOTA(1,4,7-triazacyclononanetriacetic acid), TET_(A)(1,4,8,11-tetraazacyclotetradecanetetraacetic. acid), THT4′-(3-amino-4-methoxy-phenyl)-6,6″-bis(N′,N′-dicarboxymethyl-N-methylhydrazino)-2,2′:6′,2″-terpyridine),etc. In this regard, the reader is referred to the patent literature ofSterling Winthrop, Nycomed (including Nycomed Imaging and NycomedSalutar), Schering, Mallinckrodt, Bracco and Squibb relating tochelating agents for diagnostic metals, eg. in MR, X-ray andradiodiagnostic agents. See for example U.S. Pat. No. 4,647,447,EP-A-71564, U.S. Pat. No. 4,687,659, WO89/00557, U.S. Pat. No.4,885,363, and EP-A-232751.

Reporter

The reporter moieties in the contrast agents of the invention may be anymoiety capable of detection either directly or indirectly in an in vivodiagnostic imaging procedure, eg. moieties which emit or may be causedto emit detectable radiation (eg. by radioactive decay, fluorescenceexcitation, spin resonance excitation, etc.), moieties which affectlocal electromagnetic fields (eg. paramagnetic, superparamagnetic,ferrimagnetic or ferromagnetic species), moieties which absorb orscatter radiation energy (eg. chromophores and fluorophores), particles(including liquid containing vesicles), heavy elements and compoundsthereof, and moieties which generate a detectable substance, etc.

A very wide range of materials detectable by diagnostic imagingmodalities is known from the art and the reporter will be selectedaccording to the imaging modality to be used. Thus for example forultrasound imaging an echogenic material, or a material capable ofgenerating an echogenic material will normally be selected, for X-rayimaging the reporter will generally be or contain a heavy atom (eg. ofatomic weight 38 or above), for MR imaging the reporter will either be anon zero nuclear spin isotope (such as ¹⁹F) or a material havingunpaired electron spins and hence paramagnetic, superparamagnetic,ferrimagnetic or ferromagnetic properties, for light imaging thereporter will be a light scatterer (eg. a coloured or uncolouredparticle), a light absorber or a light emitter, for magnetometricimaging the reporter will have detectable magnetic properties, forelectrical impedance imaging the reporter will affect electricalimpedance and for scintigraphy, SPECT, PET etc. the reporter will be aradionuclide.

Examples of suitable reporters are widely known from the diagnosticimaging literature, eg. magnetic iron oxide particles, X-ray contrastagent containing vesicles, chelated paramagnetic metals (such as Gd, Dy,Mn, Fe etc.). See for example U.S. Pat. No. 4,647,447, PCT/GB97/00067,U.S. Pat. No. 4,863,715, U.S. Pat. No. 4,770,183, WO96/09840,WO85/02772, WO92/17212, PCT/GB97/00459, EP-A-554213, U.S. Pat. No.5,228,446, WO91/15243, WO93/05818, WO96/23524, WO96/17628, U.S. Pat. No.5,387,080, WO95/26205, GB9624918.0, etc.

Particularly preferred as reporters are: chelated paramagnetic metalions such as Gd, Dy, Fe, and Mn, especially when chelated by macrocyclicchelant groups (eg. tetraazacyclododecane chelants such as DOTA, DO3A,HP-D03A and analogues thereof) or by linker chelant groups such as DTPA,DTPA-BMA, EDTA, DPDP, etc; metal radionuclide such as ⁹⁰Y, ^(99m)Tc,¹¹¹In, ⁴⁷Sc, ⁶⁷/Ga, ⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸Re, ¹⁷⁷Lu,¹⁹⁹Au, ²⁰³Pb and ¹⁴¹Ce; superparamagnetic iron oxide crystals;chromophores and fluorophores having absorption and/or emission maximain the range 300-1400 nm, especially 600 nm to 1200 nm, in particular650 to 1000 nm; chelated heavy metal cluster ions (eg. W or Mopolyoxoanions or the sulphur or mixed oxygen/sulphur analogs);covalently bonded non-metal atoms which are either high atomic number(eg. iodine) or are radioactive, eg ¹²³I, ¹³¹I, etc. atoms; iodinatedcompound containing vesicles; etc.

Stated generally, the reporter may be (1) a chelatable metal orpolyatomic metal-containing ion (ie. TcO, etc), where the metal is ahigh atomic number metal (eg. atomic number greater than 37), aparamagentic species (eg. a transition metal or lanthanide), or aradioactive isotope, (2) a covalently bound non-metal species which isan unpaired electron site (eg. an oxygen or carbon in a persistant freeradical), a high atomic number non-metal, or a radioisotope, (3) apolyatomic cluster or crystal containing high atomic number atoms,displaying cooperative magnetic behaviour (eg. superparamagnetism,ferrimagnetism or ferromagnetism) or containing radionuclides, (4) achromophore (by which term species which are fluorescent orphosphorescent are included), eg. an inorganic or organic structure,particularly a complexed metal ion or an organic group having anextensive delocalized electron system, or (5) a structure or grouphaving electrical impedance varying characteristics, eg. by virtue of anextensive delocalized electron system.

Examples of particular preferred reporter groups are described in moredetail below.

Chelated metal reporters: metal radionuclides, paramagnetic metal ions,fluorescent metal ions, heavy metal ions and cluster ions

Preferred metal radionuclides include ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ⁴⁷SC, ⁶⁷Ga,⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸Re, ¹⁷⁷Lu, ¹⁹⁹Au, ²⁰³Pb and¹⁴¹Ce.

Preferred paramagnetic metal ions include ions of transition andlanthanide metals (eg. metals having atomic numbers of 6 to 9, 21-29,42, 43, 44, or 57-71), in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu,La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu,especially of Mn, Cr, Fe, Gd and Dy, more especially Gd.

Preferred fluorescent metal ions include lanthanides, in particular La,Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. Eu isespecially preferred.

Preferred heavy metal-containing reporters may include atoms of Mo, Bi,Si, and W, and in particular may be polyatomic cluster ions (eg. Bicompounds and W and Mo oxides) as described in WO91/14460, WO92/17215,WO96/40287, and WO96/22914.

The metal ions are desirably chelated by chelant groups on the linkermoiety or in or on a particle, (eg. a vesicle or a porous or non-porousinorganic or organic solid), in particular linear, macrocyclic,terpyridine and N₂S₂ chelants, such as for example DTPA, DTPA-BMA, EDTA,D03A, TMT. Further examples of suitable chelant groups are disclosed inU.S. Pat. No. 4,647,447, WO89/00557, U.S. Pat. No. 5,367,080, U.S. Pat.No. 5,364,613, etc.

The linker moiety or the particle may contain one or more such chelantgroups, if desired metallated by more than one metal species (eg. so asto provide reporters detectable in different imaging modalities).

Particularly where the metal is non-radioactive, it is preferred that apolychelant linker or particulate reporter be used.

A chelant or chelating group as referred to herein may comprise theresidue of one or more of a wide variety of chelating agents that cancomplex a metal ion or a polyatomic ion (eg. TcO).

As is well known, a chelating agent is a compound containing donor atomsthat can combine by coordinate bonding with a metal atom to form acyclic structure called a chelation complex or chelate. This class ofcompounds is described in the Kirk-Othmer Encyclopedia of ChemicalTechnology, Vol. 5, 339-368.

The residue of a suitable chelating agent can be selected frompolyphosphates, such as sodium tripolyphosphate and hexametaphosphoricacid; aminocarboxylic acids, such as ethylenediaminetetraacetic acid,N-(2-hydroxy)ethylenediaminetriacetic acid, nitrilotriacetic acid,N,N-di(2-hydroxyethyl)glycine, ethylenebis(hydroxyphenylglycine) anddiethylenetriamine pentacetic acid; 1,3-diketones, such asacetylacetone, trifluoroacetylacetone, and thenoyltrifluoroacetone;hydroxycarboxylic acids, such as tartaric acid, citric acid, gluconicacid, and 5-sulfosalicyclic acid; polyamines, such as ethylenediamine,diethylenetriamine, triethylenetetraamine, and triaminotriethylamine;aminoalcohols, such as triethanolamine andN-(2-hydroxyethyl)ethylenediamine; aromatic heterocyclic bases, such as2,2′-diimidazole, picoline amine, dipicoline amine and1,10-phenanthroline; phenols, such as salicylaldehyde,disulfopyrocatechol, and chromotropic acid; aminophenols, such as8-hydroxyquinoline and oximesulfonic acid; oximes, such asdimethylglyoxime and salicylaldoxime; peptides containing proximalchelating functionality such as polycysteine, polyhistidine,polyaspartic acid, polyglutamic acid, or combinations of such aminoacids; Schiff bases, such as disalicylaldehyde 1,2-propylenediimine;tetrapyrroles, such as tetraphenylporphin and phthalocyanine; sulfurcompounds, such as toluenedithiol, meso-2,3-dimercaptosuccinic acid,dimercaptopropanol, thioglycolic acid, potassium ethyl xanthate, sodiumdiethyldithiocarbamate, dithizone, diethyl dithiophosphoric acid, andthiourea; synthetic macrocyclic compounds, such as dibenzo[18]crown-6,(CH₃)₆-[14]-4,11]-diene-N₄, and (2.2.2-cryptate); phosphonic acids, suchas nitrilotrimethylene-phosphonic acid,ethylenediaminetetra(methylenephosphonic acid), andhydroxyethylidenediphosphonic acid, or combinations of two or more ofthe above agents. The residue of a suitable chelating agent preferablycomprises a polycarboxylic acid group and preferred examples include:ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA);N,N,N′,N″,N″-diethylene-triaminepentaacetic acid (DTPA);1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA);1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid (DO3A);1-oxa-4,7,10-triazacyclododecane-N,N′,N′″-triacetic acid (OTTA);trans(1,2)-cyclohexanodiethylene-triaminepentaacetic acid (CDTPA).

Other suitable residues of chelating agents comprise proteins modifiedfor the chelation of metals such as technetium and rhenium as describedin U.S. Pat. No. 5,078,985, the disclosure of which is herebyincorporated by reference.

Suitable residues of chelating agents may also derive from N3S and N2S2containing compounds, as for example, those disclosed in U.S. Pat. Nos.4,444,690; 4,670,545; 4,673,562; 4,897,255; 4,965,392; 4,980,147;4,988,496; 5,021,556 and 5,075,099.

Other suitable residues of chelating are described in PCT/US91/08253,the disclosure of which is hereby incorporated by reference.

Preferred chelating groups are selected from the group consisting of2-amiomethylpyridine, iminoacetic acid, iminodiacetic acid,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA), carbonyliminodiacetic acid, methyleneiminoacetic acid,methyleneiminodiacetic acid, ethylenethioethylene-iminoacetic acid,ethylenethioethyleneiminodiacetic acid, TMT, a terpyridinyl group, achelating agent comprising a terpyridyl group and a carboxymethylaminogroup, or a salt of any of the foregoing acids. Especially preferredchelating groups are DTPA, DTPA-BMA, DPDP, TMT, DOTA and HPDO3A.

Representative chelating groups are also described in U.S. Pat. No.5,559,214 A, WO 9526754, WO 9408624, WO 9409056, WO 9429333, WO 9408624,WO 9408629 A1, WO 9413327 A1 and WO 9412216 A1.

Methods for metallating any chelating agents present are within thelevel of skill in the art. Metals can be incorporated into a chelantmoiety by any one of three general methods: direct incorporation,template synthesis and/or transmetallation. Direct incorporation ispreferred.

Thus it is desirable that the metal ion be easily complexed to thechelating agent, for example, by merely exposing or mixing an aqueoussolution of the chelating agent-containing moiety with a metal salt inan aqueous solution preferably having a pH in the range of about 4 toabout 11. The salt can be any salt, but preferably the salt is a watersoluble salt of the metal such as a halogen salt, and more preferablysuch salts are selected so as not to interfere with the binding of themetal ion with the chelating agent. The chelating agent-containingmoiety is preferably in aqueous solution at a pH of between about 5 andabout 9, more preferably between pH about 6 to about 8. The chelatingagent-containing moiety can be mixed with buffer salts such as citrate,acetate, phosphate and borate to produce the optimum pH. Preferably, thebuffer salts are selected so as not to interfere with the subsequentbinding of the metal ion to the chelating agent.

In diagnostic imaging, the vector-linker-reporter (VLR) constructpreferably contains a ratio of metal radionuclide ion to chelating agentthat is effective in such diagnostic imaging applications. In preferredembodiments, the mole ratio of metal ion per chelating agent is fromabout 1:1,000 to about 1:1.

In radiotherapeutic applications, the VLR preferably contains a ratio ofmetal radionuclide ion to chelating agent that is effective in suchtherapeutic applications. In preferred embodiments, the mole ratio ofmetal ion per chelating agent is from about 1:100 to about 1:1. Theradionuclide can be selected, for example, from radioisotopes of Sc, Fe,Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Ru, Sn, Sr, Sm, Lu, Sb, W, Re,Po, Ta and Tl. Preferred radionuclides include ⁴⁴Sc, ⁶⁴Cu, ⁶⁷Cu, ²¹²Pb,⁶⁸Ga, ⁹⁰Y, ¹⁵³Sm, ²¹²Bi, ¹⁸⁶Re and ¹⁸⁸Re. Of these, especially preferredis ⁹⁰Y. These radioisotopes can be atomic or preferably ionic.

The following isotopes or isotope pairs can be used for both imaging andtherapy without having to change the radiolabeling methodology orchelator: ⁴⁷Sc₂₁; ¹⁴¹Ce₅₈; ¹⁸⁸Re₇₅; ¹⁷⁷Lu₇₁; ¹⁹⁹Au₇₉; ⁴⁷Sc₂₁; ¹³¹I₅₃;⁶⁷Cu₂₉; ¹³¹I₅₃ and ¹²³I₅₃; ¹⁸⁸Re₇₅ and ^(99m)Tc₄₃; ⁹⁰Y₃₉ and ⁸⁷Y₃₉;⁴⁷Sc₂₁ and ⁴⁴Sc₂₁; ⁹⁰Y₃₉ and ¹²³I_(53;) ¹⁴⁶Sm₆₂ and ¹⁵³Sm₆₂; and ⁹⁰Y₃₉and ¹¹¹In₄₉.

Where the linker moiety contains a single chelant, that chelant may beattached directly to the vector moiety, eg. via one of the metalcoordinating groups of the chelant which may form an ester, amide,thioester or thioamide bond with an amine, thiol or hydroxyl group onthe vector. Alternatively the vector and chelant may be directly linkedvia a functionality attached to the chelant backbone, eg. aCH₂-phenyl-NCS group attached to a ring carbon of DOTA as proposed byMeares et al. in JACS 110:6266-6267(1988), or indirectly via a homo orhetero-bifunctional linker, eg. a bis amine, bis epoxide, diol, diacid,difunctionalised PEG, etc. In that event, the bifunctional linker willconveniently provide a chain of 1 to 200, preferably 3 to 30 atomsbetween vector and chelant residue.

Where the linker moiety contains a plurality of chelant groups, thelinker preferably is or contains portions of formula

where Ch is a chelant moiety and Li is a linker backbone component, ie.the linker preferably has pendant chelants, in-backbone chelants orterminal chelants or a combination thereof. The pendant and in-backbonepolymeric structures may be branched but more preferably are linear andthe repeat units (LiCh) or other repeat units in the polymer may havein-backbone or pendant biodistribution modifying groups, eg.polyalkylene groups as in WO94/08629, WO94/09056, and WO96/20754. Theterminal chelant structures Li(Ch)_(n), which may be dendritic polymersas in WO93/06868, may have biodistribution modifying groups attached totermini not occupied by chelants and may have biodegradation enhancingsites within the linker structure as in WO95/28966.

The chelant moieties within the polychelant linker may be attached viabackbone functionalization of the chelant or by utilization of one ormore of the metal co-ordinating groups of the chelant or by amide orether bond formation between acid chelant and an amine or hydroxylcarrying linker backbone, eg. as in polylysine-polyDTPA,polylysine-polyDOTA and in the so-called magnifier polychelants, ofPCT/EP96/00565. Such polychelant linkers may be conjugated to one ormore vector groups either directly (eg. utilizing amine, acid orhydroxyl groups in the polychelant linker) or via a bifunctional linkercompound as discussed above for monochelant linkers.

Where the chelated species is carried by a particulate (or molecularaggregate, eg. vesicular) linker, the chelate may for example be anunattached mono or polychelate (such as Gd DTPA-BMA or Gd HP-DO3A)enclosed within the particle or it may be a mono or polychelateconjugated to the particle either by covalent bonding or by interactionof an anchor group (eg. a lipophilic group) on the mono/polychelate withthe membrane of a vesicle (see for example PCT/GB95/02378).

Non-metal Atomic Reporters

Preferred non-metal atomic reporters include radioisotopes such as ¹²³Iand ¹³¹I as well as non zero nuclear spin atoms such as ¹⁹F, and heavyatoms such as I.

Such reporters, preferably a plurality thereof, eg. 2 to 200, may becovalently bonded to a linker backbone, either directly usingconventional chemical synthesis techniques or via a supporting group,eg. a triiodophenyl group.

In an embodiment of this invention, the use of radioisotopes of iodineis specifically contemplated. For example, if the vector or linker iscomprised of substituents that can be chemically substituted by iodinein a covalent bond forming reaction, such as, for example, substituentscontaining hydroxyphenyl functionality, such substituents can be labeledby methods well known in the art with a radioisotope of iodine. Theiodine species can be used in therapeutic and diagnostic imagingapplications. While, at the same time, a metal in a chelating agent onthe same vector-linker can also be used in either therapeutic ordiagnostic imaging applications.

As with the metal chelants discussed above, such metal atomic reportersmay be linked to the linker or carried in or on a particulate-linker,eg. in a vesicle (see WO95/26205 and GB9624918.0).

Linkers of the type described above in connection with the metalreporters may be used for non-metal atomic reporters with the non-metalatomic reporter or groups carrying such reporters taking the place ofsome or all of the chelant groups.

Organic Chromophoric or Fluorophoric Reporters

Preferred organic chromophoric and fluorophoric reporters include groupshaving an extensive delocalized electron system, eg. cyanines,merocyanines, phthalocyanines, naphthalocyanines, triphenylmethines,porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconiumdyes, azulenium dyes, indoanilines, benzophenoxazinium dyes,benzothiaphenothiazinium dyes, anthraquinones, napthoquinones,indathrenes, phthaloylacridones, trisphenoquinones, azo dyes,intramolecular and intermolecular charge-transfer dyes and dyecomplexes, tropones, tetrazines, bis(dithiolene) complexes,bis(benzene-dithiolate) complexes, iodoaniline dyes, bis(S,O-dithiolene)complexes, etc. Examples of suitable organic or metallated organicchromophores may be found in “Topics in Applied Chemistry: Infraredabsorbing dyes” Ed. M. Matsuoka, Plenum, N.Y. 1990, “Topics in AppliedChemistry: The Chemistry and Application of Dyes”, Waring et al.,Plenum, N.Y., 1990, “Handbook of Fluorescent Probes and ResearchChemicals” Haugland, Molecular Probes Inc, 1996, DE-A-4445065,DE-A-4326466, JP-A-3/228046, Narayanan et al. J. Org. Chem. 60:2391-2395 (1995), Lipowska et al. Heterocyclic Comm. 1: 427-430 (1995),Fabian et al. Chem. Rev. 92: 1197 (1992), WO96/23525, Strekowska et al.J. Org. Chem. 57: 4578-4580 (1992), WO (Axis) and WO96/17628. Particularexamples of chromophores which may be used include xylene cyanole,fluorescein, dansyl, NBD, indocyanine green, DODCI, DTDCI, DOTCI andDDTCI.

Particularly preferred are groups which have absorption maxima between600 and 1000 nm to avoid interference with haemoglobin absorption (eg.xylene cyanole).

Further such examples include:

cyanine dyes: such as heptamethinecyanine dyes, e.g. compounds 4a to 4 gTable II on page 26 of Matsuoka (supra)

4a: where Y = S, X = I, R = Et 4b: where Y = S, X = ClO₄, R = Et 4c:where Y = Cme₂, X = I, R = Me 4d: where Y = CMe₂, X = ClO₄, R = Me 4e:where Y = CH═CH, X = I, R = Et 4f: where Y = CH═CH, X = Br, R = Et 4g:where Y = CH═CH, X = ClO₄, R = Etand in Table III on page 28 of Matsuoka (supra), i.e.

where Y = O, X = I, R = Me where Y = CMe₂, X = I, R = Me where Y = S, X= Br R = Et;chalcogenopyrylomethine dyes, e.g., compounds 12 on page 31 of Matsuoka(supra), i.e.

where Y=Te, Se, O or NR;monochalcogenopyrylomethine dyes, e.g. compounds 13 on page 31, ofMatsuoka (supra) i.e.

where n=1 or 2;pyrilium dyes, e.g., compounds 14 (X=O) on page 32 of Matsuoka (supra),i.e.

where X=O, S, or Se;thiapyrilium dyes, e.g. compounds 15 on page 32, and compound I on page167 of Matsuoka (supra), i.e.

where n 1 or 2;squarylium dyes, e.g. compound 10 and Table IV on page 30 of Matsuoka(supra), i.e.

where X=CH═CH, Y=H, and R=Et,

-   X=S, Y=H, and R=Et, and-   X=CMe₂, Y=H, and R=Me,    and compound 6, page 26, of Matsuoka (supra), i.e.

where X=CH═CH, Y=H, and R=Et;croconium dyes, e.g. compound 9 and Table IV on page 30 of Matusoka(supra), i.e.

where X=CH═CH, Y=H, and R=Et,

-   X=S, Y=H, and R=Et,-   X=CMe₂, Y=H, and R=Me,    and compound 7, page 26, of Matsuoka (supra), i.e.

where X=CH═CH, Y=H, and R=Et;azulenium dyes, e.g. compound 8 on page 27 of Matsuoka (supra), i.e.

merocyanine dyes, e.g. compound 16, R=Me, on page 32 of Matsuoka(supra), i.e.

indoaniline dyes such as copper and nickel complexes of indoanilinedyes, e.g. compound 6 on page 63 of Matsuoka (supra), i.e.

where R=Et, R′=Me, M=Cu,

-   R=Et, R′=Me, M=Ni,-   R=Me, R′=H, M=Cu, or-   R=Me, R¹=H, M=Ni,    benzo[a]phenoxazinium dyes and benzo[a]phenothiazinium dyes, e.g. as    shown on page 201 of Matusoka (supra), i.e.

where X=O or S;1,4-diaminoanthraquinone(N-alkyl)-3′-thioxo-2,3-dicarboximides, e.g.compound 20, on page 41 of Matusoka (supra)

indanthrene pigments, e.g.

see compound 21 on page 41 of Matsuoka (supra);

-   2-arylamino-3,4-phthaloylacridone dyes, e.g. compound 22 on page 41    of Matsuoka (supra)

trisphenoquinone dyes, e.g. compound 23 on page 41 of Matsuoka (supra)

azo dyes, e.g. the monoazo dye, compound 2 on page 90 of Matsuoka(supra), i.e.

where X=CH═C(CN)₂, R₁=R₂=Et, R₃=R₄=H,

-   X=C(CN)═C(CN)₂, R₁=R₂=Et, R₃=R₄=H, or-   X=

and Y=C═O, R₁=R₂=Et, R₃=R₄=H, or Y=SO₂, R₁=H, R₂=CH(Me)nBu, R₃=OMe, andR₄=NHAC;azo dyes, e.g. the polyazo dye, compound 5 on page 91 of Matsuoka(supra), i.e.

intramolecular charge transfer donor-acceptor infrared dyes, e.g.compounds 6 and 7 on page 91 of Matsuoka (supra), i.e.

and

nonbenzenoid aromatic dyes, e.g. compound 8, a tropone, on page 92, ofMatsuoka (supra), i.e.

tetrazine radical dyes, e.g. compound 9 on page 92 of Matsuoka (supra),i.e.

in which, X=p-phenylene orX=p-terphenylene as well as compound 10 on page 92 of Matsuoka (supra),i.e.

in which X=p-biphenyl;cationic salts of tetrazine radical dyes, e.g. compound 11 on page 92 ofMatsuoka (supra)

in which X=p-phenylene;donor-acceptor intermolecular charge transfer dyes, e.g. CT complexes ofcompounds 13b and 14a to 14c on page 93 of Matsuoka (supra), i.e.

where X=CH═N—N(Ph)₂ in the donor and

-   a) Y=CN, Z=NO₂-   b) Y=CN, Z=H or-   a) Y=Cl, Z=NO₂ in the acceptor;    anthraquinone dyes, e.g. compounds 12 (X=S or Se) on page 38 of    Matsuoka (supra), i.e.

wherein X=S or Se and Y=tetrachloro, tetrabromo, 2,3-dicarboxylic acid,2,3-dicarboxylic anhydride, or 2,3-dicarboxylic acid N-phenyl imide;naphthoquinone dyes, e.g. compounds 2, 3, and 4 on page 37, of Matsuoka(supra), i.e.

metallated azo dyes such as azo dyes containing nickel, cobalt, copper,iron, and manganese;phthalocyanine dyes, e.g. compound 1 in Table II on page 51 of Matsuoka(supra), e.g.

naphthalocyanine dyes, e.g. compound 3 in Table II on page 51 ofMatsuoka (supra), e.g.

metal phthalocyanines such as phthalocyanines containing aluminum,silicon, nickel, zinc, lead, cadmium, magnesium, vanadium, cobalt,copper, and iron, e.g. compound 1 in Table III on page 52 of Matsuoka(supra), e.g.

in which, for example, M=Mg;metal naphthalocyanines such as naphthalocyanines containing aluminum,zinc, cobalt, magnesium, cadmium, silicon, nickel, vanadium, lead,copper, and iron, see compound 3 in Table III on page 52 of Matsuoka(supra), e.g.

in which, for example, M=Mg;bis(dithiolene) metal complexes comprising a metal ion such as nickel,cobalt, copper, and iron coordinated to four sulfur atoms in abis(S,S′-bidentate) ligand complex, e.g. see Table I on page 59 ofMatsuoka (supra)

where

-   R₁=R₂=CF₃, M=Ni,-   R₁=R₂=phenyl, M=Pd,-   R₁=R₂=phenyl, M=Pt,-   R₁=C4 to C10 alkyl, R₂=H, M=Ni,-   R₁=C4 to C10 alkyl, R₂=H, M=Pd,-   R₁=C4 to C10 alkyl, R₂=H, M=Pt,-   R₁=R₂=phenyl, M=Ni,-   R₁=R₂=p-CH₃-phenyl, M=Ni,-   R₁=R₂=p-CH₃O-phenyl, M=Ni,-   R₁=R₂=p-Cl-phenyl, M=Ni,-   R₁=R₂=p-CF₃-phenyl, M=Ni,-   R₁=R₂=3,4,-diCl-phenyl, M=Ni,-   R₁=R₂=o-Cl-phenyl, M=Ni,-   R₁=R₂=o-Br-phenyl, M=Ni,-   R₁=R₂=3,4,-diCl-phenyl, M=Ni,-   R₁=R₂=p-CH₃, M=Ni,-   R₁=R₂=2-thienyl, M=Ni,-   R₁=p-(CH₃)₂N-phenyl, R₂=phenyl, M=Ni, and-   R₁=p-(CH₃)₂N-phenyl, R₂=p-H₂N-phenyl, M=Ni;    bis(benzenedithiolate) metal complexes comprising a metal ion such    as nickel, cobalt, copper, and iron coordinated to four sulfur atoms    in a ligand complex, e.g. see Table III on page 62 of Matsuoka    (supra), i.e.

where

-   X=tetramethyl, M=Ni,-   X=4,5-dimethyl, M=Ni,-   X=4-methyl, M=Ni,-   X=tetrachloro, M=Ni,-   X=H, M=Ni,-   X=4-methyl, M=Co,-   X=4-methyl, M=Cu, and-   X=4-methyl, M=Fe;    N,O-bidentate indoaniline dyes comprising a metal ion such as    nickel, cobalt, copper, and iron coordinated to two nitrogen and two    oxygen atoms of two N,O-bidentate indoaniline ligands, e.g. compound    6 in Table IV on page 63 of Matsuoka (supra), e.g.

where R=Et, R′=Me, M=Cu,

-   R=Et, R′=Me, M=Ni,-   R=Me, R′=H, M=Cu, and-   R=Me, R′=H, M=Ni,    bis(S,O-dithiolene) metal complexes comprising a metal ion such as    nickel, cobalt, copper, and iron coordinated to two sulfur atoms and    two oxygen atoms in a bis(S,O-bidentate) ligand complex, e.g. see    U.S. Pat. No. 3,806,462, e.g.

a-diimine-dithiolene complexes comprising a metal ion such as nickel,cobalt, copper, and iron coordinated to two sulfur atoms and twoimino-nitrogen atoms in a mixed S,S- and N,N-bidentate diligand complex,e.g. see Table II on page 180, second from bottom, of Matsuoka (supra)(also see Japanese patents: 62/39,682, 63/126,889 and 63/139,303), e.g.

andtris(a-diimine) complexes comprising a metal ion coordinated to sixnitrogen atoms in a triligand complex, e.g. see Table II on page 180 ofMatsuoka (supra), last compound, (also see Japanese Patents 61/20,002and 61/73,902), e.g.

Representative examples of visible dyes include fluorescein derivatives,rhodamine derivatives, coumarins, azo dyes, metalizable dyes,anthraquinone dyes, benzodifuranone dyes, polycyclic aromatic carbonyldyes, indigoid dyes, polymethine dyes, azacarbocyanine dyes, hemicyaninedyes, barbituates, diazahemicyanine dyes, stryrl dyes, diaryl carboniumdyes, triaryl carbonium dyes, phthalocyanine dyes, quinophthalone dyes,triphenodioxazine dyes, formazan dyes, phenothiazine dyes such asmethylene blue, azure A, azure B, and azure C, oxazine dyes, thiazinedyes, naphtholactam dyes, diazahemicyanine dyes, azopyridone dyes,azobenzene dyes, mordant dyes, acid dyes, basic dyes, metallized andpremetallized dyes, xanthene dyes, direct dyes, leuco dyes which can beoxidized to produce dyes with hues bathochromically shifted from thoseof the precursor leuco dyes, and other dyes such as those listed byWaring, D. R. and Hallas, G., in “The Chemistry and Application ofDyes”, Topics in Applied Chemistry, Plenum Press, New York, N.Y., 1990.Additonal dyes can be found listed in Haugland, R. P., “Handbook ofFluorescent Probes and Research Chemicals”, Sixth Edition, MolecularProbes, Inc., Eugene Oreg., 1996.

Such chormophores and fluorophores, may be covalently linked eitherdirectly to the vector or to or within a linker structure. Once againlinkers of the type described above in connection with the metalreporters may be used for organic chromophores or fluorophores with thechromophores/fluorophores taking the place of some or all of the chelantgroups.

As with the metal chelants discussed above chromophores/fluorophores maybe carried in or on a particulate linker-moieties, eg. in or on avesicle or covalently bonded to inert matrix particles that can alsofunction as a light scattering reporter.

Particulate Reporters or Linker-Reporters

The particulate reporters and linker-reporters generally fall into twocategories—those where the particle comprises a matrix or shell whichcarries or contains the reporter and those where the particle matrix isitself the reporter. Examples of the first category are: vesicles (eg.micelles and liposomes) containing a liquid or solid phase whichcontains the contrast effective reporter, eg. a chelated paramagneticmetal or radionuclide, or a water-soluble iodinated X-ray contrastagent; porous particles loaded with the reporter, eg. paramagnetic metalloaded molecular sieve particles; and solid particles, eg. of an inertbiotolerable polymer, onto which the reporter is bound or coated, eg.dye-loaded polymer particles.

Examples of the second category are: light scattering organic orinorganic particles; magnetic particles (ie. superparamagnetic,ferromagnetic or ferrimagnetic particles); and dye particles.

Preferred particulate reporters or reporter-linkers includesuperparamagnetic particles (see U.S. Pat. No. 4,770,183,PCT/GB97/00067, WO96/09840, etc.), echogenic vesicles (see WO92/17212,PCT/GB97/00459, etc.), iodine-containing vesicles (see WO95/26205 andGB9624918.0), and dye-loaded polymer particles (see WO96/23524).

The particulate reporters may have one or more vectors attached directlyor indirectly to their surfaces. Generally it will be preferred toattach a plurality (eg. 2 to 50) of vector moieties per particle.Particularly conveniently, besides the desired targeting vector, onewill also attached flow decelerating vectors to the particles, ie.vectors which have an affinity for the capillary lumen or other organsurfaces which is sufficient to slow the passage of the contrast agentthrough the capillaries or the target organ but not sufficient on itsown to immobilise the contrast agent. Such flow decelerating vectors(described for example in GB9700699.3) may moreover serve to anchor thecontrast agent once it has bound to its target site.

The means by which vector to particle attachment is achieved will dependon the nature of the particle surface. For inorganic particles, thelinkage to the particle may be for example by way of interaction betweena metal binding group (eg. a phosphate, phosphonate or oligo orpolyphosphate group) on the vector or on a linker attached to thevector. For organic (eg. polymeric) particles, vector attachment may beby way of direct covalent bonding between groups on the particle surfaceand reactive groups in the vector, eg. amide or ester bonding, or bycovalent attachment of vector and particle to a linker. Linkers of thetype discussed above in connection with chelated metal reporters may beused although in general the linkers will not be used to coupleparticles together.

For non-solid particles, eg. droplets (for example of water insolubleiodinated liquids as described in U.S. Pat. No. 5,318,767, U.S. Pat. No.5,451,393, U.S. Pat. No. 5,352,459 and U.S. Pat. No. 5,569,448) andvesicles, the linker may conveniently contain hydrophobic “anchor”groups, for example saturated or unsaturated C₁₂₋₃₀ chains, which willpenetrate the particle surface and bind vector to particle. Thus forphospholipid vesicles, the linker may serve to bind the vectorcovalently to a phospholipid compatible with the vesicle membrane.Examples of linker binding to vesicles and inorganic particles aredescribed in GB9622368.0 and PCT/GB97/00067.

Besides the vectors, other groups may be bound to the particle surface,eg. stabilisers (to prevent aggregation) and biodistribution modifierssuch as PEG. Such groups are discussed for example in PCT/GB97/00067,WO96/09840, EP-A-284549 and U.S. Pat. No. 4,904,479.

Preferably the V-L-R agents of the invention will have the receptortargeting vectors coupled directly or indirectly to a reporter, eg. withcovalently bound iodine radioisotopes, with metal chelates attacheddirectly or via an organic linker group or coupled to a particulatereporter or linker-reporter, eg. a superparamagnetic crystals(optionally coated, eg. as in PCT/GB97/00067), or a vesicle, e.g. aniodinated contrast agent containing micelle or liposome.

Put briefly, for the imaging modalities of MRI, X-ray, light imaging,nuclear imaging, magnetotomography and electrical impedance tomography,the favoured reporters may be as follows:

MRI Superparamgnetic iron oxide particles, in general having a particlesize smaller than about 80 nm. In particular iron oxides coated withvarious coating materials such as polyelectrolytes, PEG, starch andhyrolyzed starch are preferred. Paramagnetic metal substances includingboth chelates and particulate materials are also useful. Light imagingAny light imaging reporter group. The focus should be on substancesabsorbing in the near infrared range. Nuclear medicine Radioactivechelates comprising ⁹⁹Tc or ¹¹¹In as well as direct radiolabelledvectors having radiolabelled halogens substituents such as ¹²³I, ¹²⁵I,¹³¹I, ⁷⁵Br or ⁷⁷Br. Magnetotomography Superparmagnetic iron oxideparticles as described above. Electrical impedance Polyionic species,e.g. polymers tomography with ionic groups in the repeat units.

The agents of the invention may be administered to patients for imagingin amounts sufficient to yield the desired contrast with the particularimaging technique. Where the reporter is a metal, generally dosages offrom 0.001 to 5.0 mmoles of chelated imaging metal ion per kilogram ofpatient bodyweight are effective to achieve adequate contrastenhancements. For most MRI applications preferred dosages of imagingmetal ion will be in the range of from 0.02 to 1.2 mmoles/kg bodyweightwhile for X-ray applications dosages of from 0.05 to 2.0 mmoles/kg aregenerally effective to achieve X-ray attenuation. Preferred dosages formost X-ray applications are from 0.1 to 1.2 mmoles of the lanthanide orheavy metal compound/kg bodyweight. Where the reporter is aradionuclide, dosages of 0.01 to 100 mCi, preferably 0.1 to 50 mCi willnormally be sufficient per 70 kg bodyweight. Where the reporter is asuperparamagnetic particle, the dosage will normally be 0.5 to 30 mgFe/kg bodyweight.

The dosage of the compounds of the invention for therapeutic use willdepend upon the condition being treated, but in general will be of theorder of from 1 pmol/kg to 1 mmol/kg bodyweight.

The compounds of the present invention may be formulated withconventional pharmaceutical or veterinary aids, for example emulsifiers,fatty acid esters, gelling agents, stabilizers, antioxidants, osmolalityadjusting agents, buffers, pH adjusting agents, etc., and may be in aform suitable for parenteral administration, for example injection orinfusion or administration directly into the vasculature. Thus thecompounds of the present invention may be in conventional pharmaceuticaladministration forms such as solutions, suspensions and dispersions inphysiologically acceptable carrier media, for example water forinjections.

The compounds according to the invention may therefore be formulated foradministration using physiologically acceptable carriers or excipientsin a manner fully within the skill of the art. For example, thecompounds, optionally with the addition of pharmaceutically acceptableexcipients, may be suspended or dissolved in an aqueous medium, with theresulting solution or suspension then being sterilized.

Parenterally administrable forms, e.g. intravenous solutions, should besterile and free from physiologically unacceptable agents, and shouldhave low osmolality to minimize irritation or other adverse effects uponadministration, and thus the contrast medium should preferably beisotonic or slightly hypertonic. Suitable vehicles include aqueousvehicles customarily used for administering parenteral solutions such asSodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection andother solutions such as are described in Remington's PharmaceuticalSciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington:American Pharmaceutical Association (1975). The solutions can containpreservatives, antimicrobial agents, buffers and antioxidantsconventionally used for parenteral solutions, excipients and otheradditives which are compatible with the chelates and which will notinterfere with the manufacture, storage or use of products.

The agents of formula I may be therapeutically effective in thetreatment effective in the treatment of disease states as well asdetectable in in vivo imaging. Thus for example the vector on thereporter moieites may have therapeutic efficacy, eg. by virtue of theradiotherapeutic effect of a radionuclide reporter, the efficacy inphotodynamic therapy of a chromophore (or fluorophore) reporter or thechemotherapeutic effect of the vector moiety.

Use of the agents of formula I in the manufacture of therapeuticcompositions and in methods of therapeutic or prophylactic treatment ofthe human or non-human animal body are thus considered to representfurther aspects of the invention.

The present invention will now be further illustrated by way of thefollowing non-limiting examples. Unless otherwise indicated, allpercentages given are by weight.

EXAMPLE 1

Contrast Agent for MR Imaging of Angiogenesis

Compound 1

Lysine (0.1 g, 0.7 mmol) is added to a solution ofN²-[3S-hydroxy-4-(N-hydroxyamino)2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy)phenylalanine-N¹-methylamide(prepared in accordance with WO94/02447,0.3 g, 0.7 mmol) and DCC(N,N-dicyclohexylcarbodiimide) in dry DMF (N,N-dimethylformamide). Thereaction mixture is stirred at ambient temperature and is followed byTLC.

The dispersion is left overnight at +4° C. The dispersion is filteredand the solvent rotary evaporated before the substance is purified bychromatography.

Compound 2

Diethylenetriaminepentaacetic acid dianhydride (17.9 g, 50 mmol) isdissolved in dry DMF and compound 1 (0.3 g, 0.5 mmol) dissolved in dryDMF is added. The reaction mixture is stirred at elevated temperatureunder nitrogen atmosphere. The reaction is followed by TLC. The solventis rotary evaporated and the substance purified by chromatography.

Gd(III) Chelate of Compound 2

To a solution of compound 2 (0.4 g, 0.4 mmol) in water is addedgadolinium oxide Gd₂O₃ (0.1 g, 0.2 mmol) and the mixture is heated at95° C. After filtration the solution is evaporated and dried in vacuo at50° C.

EXAMPLE 2

Contrast Agent for MR Imaging of Angiogenesis

Compound 3

Lysine (0.1 g, 0.7 mmol) is added to a solution ofN-(4-octylphenyl)-3-(2-carboxyethyl)-6,7-dihydro-5H-thiazolo[3,2-a]pyrimidine-2-carboxamide(prepared in accordance with EP-A-618208, 0.3 g, 0.7 mmol) and DCC(N,N′-dicyclohexylcarbodiimide) in dry DMF (N,N-dimethylformamide). Thereaction mixture is stirred at ambient temperature and is followed byTLC. The dispersion is left overnight at +4° C. The dispersion isfiltered and the solvent rotary evaporated before the substance ispurified by chromatography.

Compound 4

Diethylenetriaminepentaacetic acid dianhydride (17.9 g, 50 mmol) isdissolved in dry DMF and compound 3 (0.3 g, 0.,5 mmol) dissolved in dryDMF is added. The reaction mixture is stirred at elevated temperatureunder nitrogen atmosphere. The reaction is followed by TLC. The solventis rotary evaporated and the substance purified by chromatography.

Gd(III) Chelate of Compound 4

To a solution of compound 4 (0.4 g, 0.4 mmol) in water is addedgadolinium oxide Gd₂O₃ (0.1 g, 0.2 mmol) and the mixture is heated at95° C. After filtration the solution is evaporated and dried in vacuo at50° C.

EXAMPLE 3

Contrast Agent for Nuclear Medicine for Detection of Angiogenesis

^(99m)Tc Chelate of Compound 2

Compound 2 from Example 1 (1 mg) is dissolved in 0.1 N NaOH. SnCl₂.2H₂O(100 μg) dissolved in 0.05 N HCl and a solution of 10-100 mCi ^(99m)Tcin the form of sodium pertechnetate in saline is added. The pH of thesolution is adjusted to pH 7-8 by addition of 0.5 M phosphate buffer (pH5) after less than one minute. The reaction is followed by TLC and thesubstance is purified by chromatography.

EXAMPLE 4

Contrast Agent for Nuclear Medicine for Detection of Angiogenesis

^(99m)Tc Chelate of Compound 4

Compound 4 from Example 2 (1 mg) is dissolved in 0.1 N NaOH. SnCl₂.2H₂O(100 μg) dissolved in 0.05 N HCl and a solution of 10-100 mCi ^(99m)Tcin the form of sodium pertechnetate in saline is added. The pH of thesolution is adjusted to pH 7-8 by addition of 0.5 M phosphate buffer (pH5) after less than one minute. The reaction is followed by TLC and thesubstance is purified by chromatography.

EXAMPLE 5

Contrast Agent for Nuclear Medicine for Detection of Angiogenesis

An aqueous solution of ¹³¹I₂(2 equivalents) and sodium perchlorate (1equivalent) is added to an aqueous solution ofN²-[3S-hydroxy-4-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N¹-methylamide(prepared in accordance with WO94/02446, 1 equivalent). The solvent isrotary evaporated and the substance is purified by chromatography.

EXAMPLE 6

Preparation of a DTPA Monoamide Gadolinium Complex Comprising a Vectorfor Targeting of VEGF Receptor for MR Detection of Angiogenesis

(Gd complex)

a) Synthesis of 6,7-dimethoxy-3H-quinazolin-4-one

A mixture of 2-amino-4,5-dimethoxybenzoic acid (9.9 mg, 0.050 mmol) andformamide (5 ml) was heated at 190° C. for 6 hours. The mixture wascooled to 80° C. and poured onto water (25 ml). Precipitated materialwas filtered off, washed with water and dried in vacuo. Yield 1.54 g(15%), brown powder. The structure was confirmed by ¹H (500 MHz) and ¹³CNMR (125 MHz) analysis.

b) Synthesis of 4-chloro-6,7-dimethoxyquinazoline

A suspension of compound from a) (1.03 g, 5.00 mmol) inphosphorousoxychloride (20 ml) was refluxed for 3 hours. The darksolution was concentrated and the residue was taken up in ethyl acetate.The organic phase was washed with saturated sodium bicarbonate solutionand dried (MgSO₄). The solution was filtered through a short silicacolumn and concentrated to give 392 mg (35%) of off-white material. ¹HNMR (300 MHz) and ¹³C NMR (75 MHz) spectra were in accordance with thestructure.

c) Synthesis of [4-(6,7-dimethoxy-quinazolin-4-ylamino)-phenyl]aceticacid

A mixture of compound from b) (112 mg, 0.500 mmol) and4-aminophenylacetic acid (76 mg, 0.50 mmol) in 2-propanol (8 ml) wasrefluxed for 3 hours. The reaction mixture was cooled and precipitatedmaterial was isolated, washed with 2-propanol and dried in vacuo. Yield183 mg (97%), pale yellow solid material. The structure was verified by¹H NMR (500 MHz) and ¹³C NMR (125 MHz) analysis. Furthercharacterisation was carried out using MALDI mass spectrometry(α-cyano-4-hydroxycinnamic acid matrix), giving m/z for [MH]⁺ at 341,expected 340.

d) Synthesis of t-butyl(6-{2-[4-(6,7-dimethoxy-quinazolin-4-ylamino)phenyl]acetylamino}hexyl)carbamate

To a suspension of compound from c) (38 mg, 0.10 mmol) andN-Boc-1,6-diaminohexane hydrochloride (25 mg, 0.10 mmol) in DMF (2.0 ml)was added N,N-diisopropylethyl-amine (34 ml, 0.20 mmol). To the clearsolution was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidhydrochloride (19 mg, 0.10 mmol) and 1-hydroxybenzo-triazole (15 mg,0.10 mmol). The reaction mixture was stirred at room temperatureovernight and then poured onto 25 ml of water containing sodiumcarbonate (2.5 g) and sodium chloride (4.0 g). Organic material wasextracted into chloroform and the organic phase was washed with waterand dried (Na₂SO₄). The solution was filtered and concentrated. Theproduct was purified by column chromatography (silica,chloroform/methanol/acetic acid 85:10:5) and finally lyophilised fromacetic acid. Yield 54 mg (90%), yellow-white solid material (acetate).The product was characterised by MALDI mass spectrometry(α-cyano-4-hydroxycinnamic acid matrix), giving m/z for [MH]⁺ at 539 asexpected. Further characterisation was carried out using ¹H (500 MHz)and ¹³C (125 MHz) NMR spectroscopy.

e) Synthesis ofN-(6-aminohexyl)-[4-(6,7-dimethoxy-quinazolin-4-ylamino)phenyl]acetamidehydrochloride

Compound from d) (27 mg, 0.050 mmol) was dissolved in dioxane (3 ml) bygentle heating. To the solution was added 4 N HCl in dioxane (0.5 ml).The reaction mixture was stirred overnight and concentrated in vacuo togive a quantitative yield of the title compound. Characterisation wascarried out using MALDI mass spectrometry (α-cyano-4-hydroxycinnamicacid matrix), giving m/z for [MH]⁺ at 439 as expected. Furthercharacterisation was carried out using analytical HPLC (column Vydac218TP54, gradient 12-24% B over 20 min, A=water/0.1% TFA,B=acetonitrile/0.1% TFA, flow rate 1.0 ml/min) giving a single productpeak with retention time 13.0 min detected at 340 nm. Characterisationwas also carried out by means of NMR spectroscopy, giving ¹H (500 MHz)and ¹³C (125 MHz) spectra in accordance with the structure.

f) Synthesis of a DTPA Monoamide Derivative for Gadolinium Chelation(Structure Shown Above)

N,N-Diisopropylethylamine (17 μl, 0.10 mmol) was added to a suspensionof compound from e) (0.05 mmol) and DTPA-anhydride (179 mg, 0.500 mmol)in DMF (5 ml). The mixture was stirred at room temperature for 2 hoursand concentrated in vacuo. HPLC analysis (column Vydac 218TP54, gradient16-28% B over 20 minutes, A=water/0.1% TFA, B=acetonitrile/0.1% TFA,flow rate 1.0 ml/min) gave a product peak at 7.9 min shown by LC-MS(ESI) to correspond to the title compound (m/z for [MH]⁺ at 813,expected 814). The product was purified by preparative HPLC (columnVydac 218TP1022, gradient 16-28% B over 60 min, A water/0.1% TFA,B=acetonitrile/0.1% TFA, flow rate 10.0 ml/min, detection at 254 nm)giving a yield of 6.7 mg of purified material. Analytical HPLC analysisof purified material showed a shift in retention time to 5.6 min(analytical conditions as described above), shown by MALDI massspectrometry to correspond to the iron complex, giving m/z at 870 forthe complex and 816 for the free ligand.

g) Preparation of the Gadolinium Complex of Compound from f)

Compound from f) (0.1 mg) was dissolved in an aqueous solution ofgadolinium trichloride (conc 2 mg/ml, 0.1 ml). The mixture was stirredovernight. Quantitative conversion to the gadolinium complex wasverified by MALDI mass spectrometry (α-cyano-4-hydroxycinnamic acidmatrix), giving m/z peaks at 970, 992 and 1014 for the gadoliniumcomplex (gadolinium, gadolinium/sodium and gadolinium/disodium,respectively) and at 816/838 corresponding to the free ligand/sodiumcomplex. No trace of the iron complex could be detected.

EXAMPLE 7

Preparation of a DTPA Bisamide Gadolinium Complex Comprising a Vectorfor Targeting of VEGF Receptor for MR Detection of Angiogenesis

(Gd complex)

a) Synthesis of a DTPA Bisamide Derivative for Gadolinium Chelation(Structure Shown Above)

Analytical HPLC of the reaction mixture in Example 6f) gave also a peakat 16.8 min that was shown by LC-MS (EIS) analysis to correspond to theDTPA bisamide shown above, giving m/z at 1233 for [MH]⁺ as expected and616.6 as expected for [MH₂]²⁺. The product was purified by preparativeHPLC (conditions as described in Example 6f)) to give 14 mg of purematerial after lyophilisation. Analytical HPLC analysis of the purifiedmaterial showed a shift in retention time from 16.8 min (in the crudemixture) to 11.1 min due to formation of the iron complex duringpurification, as verified by MALDI mass spectrometry(α-cyano-4-hydroxy-cinnamic acid matrix) giving m/z at 1291 for the ironcomplex and 1237 for the free ligand.

b) Preparation of the Gadolinium Complex of Compound from a)

The compound from a) was treated with an of gadolinium trichloride asdescribed in Example 6g). After 2 hours reaction time MALDI massspectrometry showed conversion to the gadolinium complex, giving m/z at1391 for the gadolinium complex and 1235 for the free ligand.

EXAMPLE 8Carboxymethyl-[2-(carboxymethyl-{2-[carboxymethyl-({2-[3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionylamino]-ethylcarbamoyl}-methyl)-amino]-ethyl}-amino)-ethyl]-amino}-aceticacid (12)

a) 4-Methyl-isophthalic acid (2)

To THF (130 ml) solution of 3-bromo-4-methyl-benzoic acid (5.0 g, 23.25mmol) under nitrogen and cooled to −78° C. (dryice/methanol) was addedMeMgBr in ether (3.0 M, 8.5 ml, 25.57 mmol) at such a rate that thetemperature did not exceed −75° C. The temperature was then allowed torise to −60° C. and after gas evolution had ceased, the solution wascooled again to −78° C. BuLi in hexane (1.6 M, 29.06 ml, 46.50 mmol) wasthen added dropwise such that the temperature did not rise above −75° C.The mixture was then stirred at this temperature for 15 minutes beforecrushed dryice (4.4 g, 100 mmol) was added. The precipitate wasvigorously stirred as the temperature was allowed to freely rise toambient temperature. The mixture was made acidic using 6 N HCl and thesolid material collected filteration, washed with diethyl ether anddried. Recrystallisation from water afforded off-white pure compound(81%) m.p. 296-298° C. (sublimed). NMR conforms to expected structure.

b) 4-Methyl-isophthalic methyl ester (4)

A mixture of compound (2) (2.83 g, 15.71 mmol), thionyl chloride (50 ml)and DMF (3 drops) was heated at reflux for 2 hours. After cooling toroom temperature, excess thionyl chloride was removed under reducedpressure (rotary evaporator). The dark oil which was obtained wasdissolved in carbon tetrachloride (30 ml), treated with pyridine (1 ml,12.43 mmol) and methanol (20 ml) and stirred at ambient temperature for2 hours. The solvents were evaporated and the residue purified by flashchromatography: silica, hexane/EtOAc (9:1).

c) 4-Bromoethyl-isophthalic acid dimethylester (5)

A mixture of (4) (0.96 g, 4.61 mmol), dibenzoylperoxide (56 mg, 0.23mmol) and N-bromosuccinimide (NBS) (0.82 g, 4.61 mmol) in carbontetrachloride (20 ml) was heated at reflux for 20 hours. After coolingto room temperature and filteration the solvent was evaporated to give ayellow oil. Flash chromatograph: silica, hexane/EtOAc (7:3) afforded thepure compound.

d)3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carboxylicacid methyl ester (6)

A solution of (5) (511 mg, 1.78 mmol) in toluene (10 ml) was treatedwith Et₃N (744 μl 5.33 mmol) and N1-pyridin-2-yl-ethane-1,2-diamine (244mg, 1.78 mmol) [prepared by treatment of 2-bromopyridine with excessethylene diamine and pyridine] and the mixture refluxed for 6 hours.After cooling to room temperature and evaporation of the solvent, theresidue was purified by flash chromatography: silica, CH₂Cl₂/acetone(3:2).

e)3-oxo-2-[2-pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carboxylicacid (7)

A methanol solution (6 ml) of (6) (301 mg, 0.97 mmol) and 1 N NaOH (3ml) was stirred at ambient temperature for 24 hours. The solution wasmade acidic using 1 M NaHSO₄ solution and the precipitated product wascollected by filteration washed thoroughly with water and dried overP₂O₅/bluegel for 24 hours.

f)3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionicacid tert-butyl ester (8)

A solution of (7) (166 mg, 0.56 mmol), N-methyl morfolin (185 μl, 1.68mmol), BOP (322 mg, 0.73 mmol) and H-β-ala-OtBu (152 mg, 0.84 mmol) inDMF (5 ml) was stirred at ambient temperature for 20 hours. The mixturewas diluted with ethyl acetate (10 ml) and then washed once each withH₂O, NaHCO₃, 10% KHSO₄ and brine (5 ml), dried (MgSO₄) and concentrated.Flash chromatography (silica, EtOAc) gave the ester (8) as a whitesolid.

g)3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionicacid (9)

A solution of the ester (8) (252 mg, 0.60 mmol), TFA (4 ml) and CH₂Cl₂(8 ml) was stirred at ambient temperature for 3 hours. The mixture wasevaporated to dryness and the residue purified by flash chromatography(silica, EtOH/NH₄OH 19:1) to provide (9) as off-white foam. NMR conformsto structure.

h){2-[3-({3-Oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carbonyl-amino)-propionylamino]-ethyl}-carbamicacid tert-butyl ester (10)

To a solution of the acid (9) (20 mg, 0.054 mmol) in DMF (2 ml) wasadded N-methyl morfolin (NMM) (16.40 mg, 0.162 mmol), BOP (castro'sreagent) 31.05 mg, 0.070 mmol) and the BOC-protected diamine (13 mg,0.081 mmol) and the mixture was stirred at ambient temperature for 20hours. After dilution with EtOAc (5 ml), the solution was washed onceeach with H₂O, sat. NaHCO₃, 10% KHSO₄ and brine. The organic phase wasdried (MgSO₄) and concentrated. Flash chromatography (silica, 1:1CH₂Cl₂/acetone) MALDI-MS; 510.59.

i)3-Oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carboxylicacid [2-(2-amino-ethylcarbamoyl)-ethyl]-amide (11)

A CH₂Cl₂ solution (3 ml) of (10) (20 mg, 0.039 mmol) and TFA (2 ml) wasstirred under ambient conditions for 4 hours. The reaction mixture wasconcentrated and the residue purified by flash chromatography (silica,19:1, CH₂Cl₂/acetone) to provide (4) as a white solid. MALDI-MS; 410.48

j)Carboxymethyl-[2-(carboxymethyl-{2-[carboxymethyl-({2-[3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionylamino]-ethylcarbamoyl}-methyl)-amino]-ethyl}-amino)-ethyl]-amino}-aceticacid (12)

A solution of (11) (15 mg, 0.36 mmol), N,N-diisopropylamine (17 μl, 0.10mmol) and DTPA-anhydride (129 mg, 0.36 mmol) in DMF (2 ml) was stirredat ambient temperature for 3 hours and concentrated in vacuo. Theresidue was purified by preparative HPLC (acetonitrile/0.1% TFA inwater).

EXAMPLE 9

Preparation of a DTPA Monoamide Gadolinium Complex Comprising a Vectorfor Targeting of bFGF Receptor for MR Detection of Angiogenesis

a) Synthesis of:{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-aceticacid tert-butyl ester

1-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-1H-imidazole (1g, 2.25 mmol) and tert-butyl bromoacetate (1 ml, 6.8 mmol) weredissolved in 15 ml acetonitrile and heated to reflux overnight. TLCshowed full conversion of the starting material. The solvent was cooled,evaporated in vacuo, and the residual oil was dissolved in chloroformand triturated with ether. The product was identified by Maldi massspectrometry, and used in the next step without further purification.

b) Synthesis of:{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-aceticacid

The compound from a) (500 mg, 1 mmol) was dissolved in 2 mldichloromethane and cooled in an icebath. 2 ml trifluoroacetic acid wasadded, the icebath was removed and the reaction mixture was stirred for1 hour. TLC showed full conversion of the starting material. The solventwas removed in vacuo, and the product used in the next step withoutfurther purification.

c) Synthesis of:[6-(2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetylamino)-hexyl]-carbamicacid tert-butyl ester

The product from b) was converted to c) by the procedure described forExample 6 d), and the product was purified by flash chromatography.

d) Synthesis of:N-(6-amino-hexyl)-2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetamide

The product from c) was converted to d) by the procedure described forExample 6 e). The product was used without further purification.

e) Synthesis of:((2-{[2-(bis-carboxymethyl-amino)-ethyl]-carboxymethyl-amino}-ethyl)-{[6-(2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetylamino)-hexylcarbamoyl]-methyl}-amino)-aceticacid

The product from d) was converted to e) by the procedure described forExample 6 f). Purification was performed by preparative HPLC asdescribed in Example 6 f).

f) Preparation of the gadolinium complex of compound e) was performed bythe procedure described in Example 6 g).

1. A composition of matter of formula IV-L-R  (Formula I) wherein L is a linker moiety or a bond; R is a moietycomprising one to ten detectable labels; and V is selected from the listof non-peptidic organic groups consisting of:N²-[3S-hydroxy-4-(N-hydroxyamino)2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy)phenylalanine-N¹-methylamide,N-(4-octylphenyl)-3-(2-carboxyethyl)-6,7-dihydro-5H-thiazolo[3,2-a]pyrimidine-2-carboxamide,N²-[3S-hydroxy-4-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N¹-methylamide,N-(6-aminohexyl)-[4-(6,7-dimethoxy-quinazolin-4-ylamino)phenyl]acetamidehydrochloride,3-Oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carboxylicacid [2-(2-amino-ethylcarbamoyl)-ethyl]-amide, andN-(6-amino-hexyl)-2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetamide.2. A composition of matter of claim 1 labelled with Gd(III), ^(99m)Tc or¹¹¹In chelates of DTPA.
 3. A composition of matter as claimed in claim 1wherein R is a radionuclide.
 4. A composition of matter as claimed inclaim 3 wherein R is an iodine or metal radionuclide.
 5. Apharmaceutical composition comprising a composition of matter of formulaI as defined in claim 1 together with at least one pharmaceuticallyacceptable carrier or excipient.
 6. A method of generating an image ofan animate human or non-human animal subject involving administering acontrast agent to said subject and generating an image of at least apart of said subject to which said contrast agent has distributed,characterised in that as said contrast agent is used as a composition ofmatter of formula I as defined in claim
 1. 7. A process for thepreparation of a composition of a matter of formula IV-L-R of claim 1, said process comprising conjugating moiety V withmoiety R via the linker or bond L.